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Affymetrix GeneChips are widely used to assess transcriptional changes in animal models and are increasingly applied for diagnostic and therapeutic evaluations in clinical settings. In many instances, an intensity error model is chosen as the technique to convert fluorescent probe intensities into expression summaries for these Affymetrix GeneChips. However, there are currently only two published reports on the precision and accuracy of this intensity error model, one of which employs a sample of known transcript concentrations for only 14 RNA species. The other report did not employ a known sample to support its conclusions. Recently a wholly defined control dataset has become available that has known RNA concentrations for 3860 individual cRNAs. In the initial publication, this dataset was used to evaluate over 150 analytical techniques, primarily using receiver operating characteristic curves. In this report, we evaluate the ability of an intensity error model to detect the presence and differential expression of transcripts in this control dataset. Initial results show that this intensity error model, using default parameters, identifies 56% of the true positives at a false-discovery rate of 0.05. In addition, this error model’s ability to detect a monotonic titration response is evaluated. This evaluation is based on Affymetrix Cel files derived from RNA sample titrations prepared by the Microarray Quality Control project. This abstract has been reviewed by the NHEERL, US EPA, and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Data sharing for proteomics research is important for collaboration in large-scale proteomics research projects.
Abstracts beginning with P are standard abstract poster presentations, F designates the new Facility Poster category, and V designates Vendor Contributed abstracts. The board numbers are followed by S (Sunday), M (Monday), and T (Tuesday), which indicates the day the author is to be present at the board.
It is also the foundation to provide public access to proteomics data for the entire research community. National Cancer Institute has implemented CPAS-Mart, a BioMart query system for the CPAS public proteomics repository.
CPAS-Mart is based on Computational Portal and Analysis System (CPAS), a proteomics repository system developed at Fred Hutchinson Cancer Research Center. An instance of CPAS is installed at National Cancer Institute (NCI) to host data generated from Mouse Proteomics Technology initiative (MPTI) and Clinical Proteomics Technology Assessment (CPTAC) initiative. Currently, the repository provides public access to 6,176,247 peptide identifications from 244 MS-MS runs.
As a public proteomics repository, we have explored options to improve data-query and filtering mechanisms. In collaboration with the Cancer Proteomics Technologies program at NCI, we chose to implement a BioMart query-oriented management interface for CPAS. It allows data mining for scientists without extensive mass spectrometry background. BioMart is developed by the European Bioinformatics Institute (EBI) and Cold Spring Harbor Laboratory (CSHL). It simplifies the task of creation and maintenance of advanced query interfaces for an existing repository. The implemented CPAS-Mart system provides increased search options on proteomics data without modifying the CPAS schema. The current CPAS-Mart schema provides access to multiple datasets with a large number of filter and attributes selections. We are currently bringing genomics data into the proteomics database with DAS server.
Driven by the rapid development of mass spectrometry (MS) instruments and methodologies, proteomics has become a remarkably powerful technology to identify proteins in complex mixtures such as cell and tissue lysates or biofluids. Computational analysis of MS data, however, is still a challenge, since vast quantities of data can be generated very quickly from proteomics experiments. Database searching and de novo sequencing are two major methods automated for peptide and protein identification. Alternatively, a sequence-tagging method hybridizes the strategies of both database searching and de novo sequencing. It infers partial sequences directly from the tandem mass spectrum and then searches the protein database to interpret the remainder. Several sequence-tagging algorithms have been developed, but the performance of these algorithms across different instrument platforms has not yet been compared. Here, we evaluate the run-time and tag qualities of DirecTag, InsPecT, GutenTag, and PepNovo algorithms. We employ datasets produced on instruments including the LTQ, LTQ-FT, QSTAR, and Orbitrap. The results demonstrate superior accuracy and efficiency in the DirecTag algorithm stemming from its high discrimination scorer and multi-threaded design.
Comprehensive proteomic analyses deal with large datasets comprising a range from very few up to thousands of spectra. Considering multiple measurements (method optimization, quality control, and long-term studies) this number multiplies. This necessitates a structured overview and an intelligent access to the different results. Software-supported database queries allow us to investigate specific aspects: different sample preparations and certain peptide- or protein-specific attributes, including biological properties. We will present a software package featuring proteomics-specific queries for MS data. As an example, the analysis of glycosylated serum proteins captured by means of different functionalized magnetic beads was performed.
Human serum, which contains a high number of glycoproteins comprising several orders of magnitude in concentration, was incubated with differentially functionalized magnetic microparticles. The released proteins were tryptically digested, and the resulting complex peptide mixtures were subjected to LC-MALDI analysis and subsequent database search. All sample- and MS-relevant information was administrated using the ProteinScape software, which likewise was used to perform the queries.
According to the specific binding preferences of the magnetic beads, different numbers of diverse glycoproteins were identified. Comparative queries, which included restrictions such as reliable scores and defined modifications (e.g., deamidation), allowed for the definition of bead-specific binding profiles. The application of software-driven queries facilitated a quick and concise overview about the different subsets. The comparison of repeated experiments revealed the reproducibility of the experimental setups. The software-supported data management allowed for quick and simple extraction of tailored and concise information from huge datasets.
Protein identification search engines such as MASCOT, X!Tandem, and ProteomicSuite Basis identify proteins with reference to a sequence database based on each original algorithm. As the varieties of identified proteins are not completely matched if their algorithms are different, for more accurate protein identification, it is important to apply multiple identification engines and compare their identification results.
We have developed the proteome comparative analysis workbench ProteomicSuite for protein identification results comparison among different identification engines. This software submits a protein identification search to pre-designated identification engines such as MASCOT with homogenized parameters at the same time and compares their results. In this poster session, we introduce the comparative method and show results using different identification engines with concrete mass measurement samples.
Proteomics today generates vast amounts of peptide and protein identification data with high accuracy. A growing issue in proteomics is data analysis, particularly analyses of protein and peptide identifications made by automated database search engines and the subsequent extraction of biologically meaningful information from mass spectrometry experiments. This poster shows the application of a new bioinformatics tool, ProteinCenter, that manages these protein and peptide lists and puts them in a biological context. The tool was developed specifically to help researchers rapidly obtain a biologically relevant overview in large-scale proteomics studies by using biological annotations from multiple resources. Within minutes, output generated by protein database search engines can be translated into biological information.
In the current study, we used ProteinCenter to analyze a quantitative proteomics study of the yeast pheromone signaling pathway.1 Using only the protein list, it was possible to deduce which cellular processes were perturbed. Among the top over-represented Gene Ontology (GO) cellular components were: site of polarized growth, mating projection, cell projection, and cellular bud neck. This correlated perfectly with the design of the experiment: the mating projection as a part of cell projection is formed by unicellular fungi in response to mating pheromone. More detailed analysis showed that mating projection is caused mainly by the mating projection tip rather than the mating projection base.
Similar analyses were performed at the level of GO Biological Process and GO Molecular Function, and a comparison with the whole yeast proteome from reference SwissProt database was made.
Result validation is a key issue for the identification and quantification of a large number of proteins. For maximizing the number of reliably identified proteins, biological and technical replicates are mandatory, as well as the combination of data from various workflows (gels, 1D, 2D LC), instruments (TOF/TOF, trap, qTOF, or FTMS) and search engines. Here, we describe a database-driven study that combines LC-based workflows on different mass spectrometers, using four search engines for protein identification. The decoy database approach for a precise determination of false-positive rates was employed.
A tryptic digest of 10,000 cells of a human cell line was used in this study. LC-MALDI TOF/TOF runs and a 2D LC ESI trap run on capillary and nano-LC columns were acquired and submitted to the proteomics database platform ProteinScape. The combined MALDI data and ESI data were searched using Mascot (Matrix Science), Phenyx (GeneBio), ProteinSolver (Bruker and Protagen) and Sequest (Thermo) against a decoy database generated from IPI-human in order to obtain one protein list across all workflows and search engines at a defined false-positive rate for protein identification. The initial separate searches from the two datasets generated eight independent peptide lists. A merged, non-redundant protein list was compiled using the ProteinExtractor algorithm in ProteinScape.
Initial evaluation of the generated data provided for the identification of ~1000 proteins derived from the 2D LC ESI approach and ~700 proteins from the 1D LC MALDI approach, each with a decoy calculated false-positive rate of 5%. The merged list of all datasets and search engines contained ~1200 proteins.
The first wave of Next Generation (“NextGen”) sequencing technologies combines molecular resolution with extremely high throughput to dramatically reduce sequencing costs and provide laboratories with “Genome Center” throughput to make discoveries and develop new assays never before imagined. However, widespread adoption of NextGen will be hindered because current bioinformatics programs do not scale; they are inefficient in data storage, processing, and memory utilization. The most popular programs typically copy and recopy data to new files many times during processing, require that all data be maintained in random access memory when running, and cannot incrementally process data. To overcome these issues, fundamental changes in data management and processing are needed.
Geospiza and the HDF Group are collaborating to develop scalable, bioinformatics technologies based on HDF5 (Hierarchical Data Format—http://www.hdfgroup.org). We call these extensible domain-specific data technologies BioHDF. BioHDF will implement data models that support DNA sequence information (reads, quality values, metadata) and results from sequence assembly and variation algorithms. BioHDF will extend HDF5 data structures and library routines with new features to support high-performance data storage and computation requirements of NextGen sequencing. BioHDF will include APIs, software tools, and a viewer based on HDFView to enable its use in the bioinformatics and research communities. Using BioHDF, researchers will be able to perform de novo sequencing, do resequencing-based SNP discovery, analyze genotyping data, and export datasets in formats ready for submission to key databases. As a programming environment, BioHDF can be easily extended to accept data from new data-collection platforms and format data for interchange with many databases. BioHDF will be delivered to the research community as an open-source technology. Geospiza will include BioHDF in its products to help laboratories deliver data to scientists working with core labs and other services.
An increasing number of algorithms for MS/MS data interpretation have been published in recent years. For each algorithm, the benefit for a given data setting was demonstrated. Clearly, the preference of utilizing a specific search engine is not only reflected by its performance but by its search time, interfaces, GUI presentation, and availability. However, the performance itself was not investigated systematically for the current search engines, as the variability of test parameters like sample organism, proteome separation technique, mass spectrometer, and search algorithm parameter is enormously high.
Here we present a systematic comparison of four algorithms (Mascot, Sequest, Phenyx, and ProteinSolver) and quantify the benefit of combining multi-search-engine results. For the test we carefully collected representative datasets from a variety of sample organisms, proteome separations, and mass spectrometer types. In total, six datasets with over 600,000 spectra served as data source. In a second step, we set up a method for comparing results based on a threshold for a given false-positive rate (ProteinExtractor algorithm combined with Decoy strategy). With this, we were able to accurate measure the performance of a search engine on a given dataset and moreover quantify the benefit of the simultaneous use of four algorithms. Additionally, the similarity of the applied algorithms is calculated on the distance matrix based on the overlap of correctly identified spectra. Preliminary results show that there is a high dependency on the dataset type for each algorithm to give the highest number of correct hits at a defined false-positive rate. However, assuming the utilized datasets in total as representative data, there is a clear performance difference between the applied algorithms. Additionally, we quantified the overall benefit of utilizing all four algorithms over utilizing the most widespread algorithm alone, to 35% higher correct identifications at a defined false-positive rate.
The Complex Carbohydrate Research Center (CCRC) of the University of Georgia offers a comprehensive service and training program for structural characterization of glycoconjugates from animal, bacterial, fungal, and plant sources. The available analyses and techniques include: glycosyl-residue composition and glycosyl linkage; identifying site(s) of attachment of N- and O-linked glycans; releasing and purifying N- and O-linked glycans; identifying the type of N-linked glycans (e.g., biantennary, triantennary, tetraantennary, high mannose, hybrid, complex); determining glycosyl residue sequence, ring size, and anomeric configuration; and identifying and determining the points of attachment of non-carbohydrate constituents such as phosphate and sulfate.
The training program includes hands-on training on structural characterization of glycoconjugates:
August 4–8, 2008—Separation and Characterization of Glycoconjugate Oligosaccharides
August 11–15, 2008—Separation and Characterization of Glycoconjugate Oligosaccharides
August 18–22, 2008—Analytical Techniques for Carbohydrates Structure Determination
August 25–27, 2008—Structural Characterization of Glycosaminoglycans (GAG)
One of the major challenges in the -omics field is the development of technologies that allow for quantitative analysis between samples. In proteomics, stable isotope approaches, such as SILAC, have been developed to address this need. Here we report a methodology that takes advantage of stable isotope labeling of glycans in cell culture for performing relative quantitative glycomics. This methodology, isotopic detection of aminosugars with glutamine (IDAWG), relies on the hexosamine biosynthetic pathway, which uses the side chain of glutamine as its sole donor source of nitrogen for aminosugars in the production of sugar nucleotides. Thus, introduction of heavy glutamine (15N) into Gln-free media allows for all aminosugars to become labeled and shifted in mass by +1 Da. Here we demonstrate that this methodology allows for rapid and nearly complete incorporation of 15N into GlcNAc, GalNAc, and sialic acids of N-linked and O-linked glycans in various mammalian cell-culture systems. Besides aiding in the assignment of structures via LC-MSn approaches, this method allows us to determine whether the glycans isolated from a sample result from cellular processes or serum glycoproteins. Importantly, this method also allows us to compare in a quantitative manner the glycans between two cell populations. Furthermore, half-life studies can be performed on glycan structures by switching a cell population from heavy to light labeling conditions and harvesting and analyzing the glycans by LC-MSn approaches at multiple time points afterwards. Thus, the IDAWG approach is an easily applied and powerful new tool in the glycomics tool-box.
Low-molecular-weight carbohydrates, such as oligosaccharides, can be difficult to separate from contaminants. Methods such as ion exchange may not be suitable due to strong hydrophilicity. Sephadex G-10 is a well-established gel filtration chromatography medium suitable for group separation of biomolecules with a molecular weight above 700 from smaller molecules such as salts, dyes, and radioactive labels. Here, we show efficient cleanup of an oligosaccharide-containing solution from salt using PD Mini-Trap G-10, a new pre-packed column format.
Despite the overwhelming success of shotgun proteomics, a number of limitations dealing with data validation and data processing are slowing down the progress of the field. The bioinformatical data interpretation and comparison of huge datasets is a tedious and extremely time-consuming process. Here, we present a software solution to speed up that process, exemplified via a comparison of proteomics data obtained from the E. coli K12 cell lines grown at 25 and 37°C.
E. coli samples were subjected to SDS-PAGE separation followed by in-gel digestion and nano-LC-MS/MS analysis on an LTQ-XL mass spectrometer. An E. coli sequence database was used for the database search, based on the X!Tandem search engine. The results of the database search were imported into ProteinCenter (www.proxeon.com) for bioinformatical comparative analysis. The data were clustered on the level of peptide-indistinguishable proteins. As a result, more than 55% of proteins were detected in both samples. The differently expressed proteins were analyzed statistically on the Gene Ontology (GO) levels.
In response to mild hypothermia, membrane protein count dropped from 22% to 16%.
A slight decrease of transport activity from 13 to 10% was also detected A significant increase of transferase, recombinase car-boxylyase, endopeptidase exonuclease, and motor activities was observed for the “cooled” cell line on the level of molecular function GO analysis. At the same time, the protein kinase transporter, ATPase, and pyrophosphatase activities decreased dramatically, especially protein histidine kinase activity. Cellular localization analysis showed overrepresentation of flagellum proteins, particularly flagellar hook proteins. Molecular function analysis indicated an increase of Momolybdopterin cofactor biosynthesis. These processes may play a central role in cell response to cold shock.
The described approach can facilitate bioinformatics data mining and show the relevant molecular mechanisms within the framework of a general cell response to cold shock.
The Center for Chemical Genomics and Translational Research (CGTR) is a joint venture between the University of Arizona and HTG, Inc., that was established in August, 2007 with initial funding from the Science Foundation, Arizona. The mission of the CGTR is to provide collaborative services and fee-based services to academic and industry partners, leveraging HTG’s quantitative Nuclease Protection Assay (qNPA) gene expression technology to address the following objectives: (1) target identification using high-density qNPA microarrays, (2) target validation using the microplate-based ArrayPlate qNPA, (3) implementation of chemical genomics programs of multiplexed drug and agrochemical discovery, including high-throughput screening and lead optimization, (4) identification and validation of predictive metabolism and safety signatures and profiling of compounds, and (5) the identification and validation of prognostic and diagnostic signatures. The last two objectives particularly exploit the unique ability of the qNPA assay to perform retrospective analysis using archived formalin-fixed, paraffin-embedded (FFPE) tissue. Current research programs are aimed toward discovery of agents useful in the production of biofuels, identification of novel drug leads, and development of FFPE-based prognostic oncology assays. Collaborative institutes include the University of Arizona BIO5 Institute and the Arizona Cancer Center.
We have compared four high-density CNV platforms—Agilent 244k, Nimblegen 385k (aCGH), Illumina Hap-370CNV, and Affymetrix SNP5.0 (snpCGH)—for use in a large breast cancer study.
This pilot study used six matched tumors and their normal DNAs and several cell lines to address the reproducibility, sensitivity, and applicability of each platform to our study.
A single platform was chosen to profile copy number on 2000 breast cancer tumors to correllate with gene expression data from the Illumina Human6 arrays. The full study is due for completion at the end of 2008.
Advances have been made regarding high-density microarray platforms, but performance continues to be limited by the assays that are run on those platforms, in which there have been few improvements. We have adapted the quantitative Nuclease Protection Assay (qNPA), marketed by HTG as a microplate-based focused array assay, to provide a high-density array platform to measure mRNA and miRNA. The advantages provided by qNPA include: (1) qNPA utilizes a lysis-only protocol, and therefore does not require extraction, reverse transcription, or amplification steps; (2) any biological sample can be tested, including archived FFPE and cells fixed in glutaraldehyde, in addition to unfixed cells, tissues, and whole organisms; (3) miRNA or siRNA can be measured without concerns regarding losses during extraction; (4) the same reagents employed in the high-density array can also be used in the focused microplate-based ArrayPlate qNPA assay, permitting rapid and cost-effective high-throughput validation of genes identified on the high-density qNPA array; (5) any region of the target gene(s) can be probed without a 3k or 5k bias; (6) the assay can be run in the absence of sample, permitting QC of every reagent and absolute validation of array performance. Data will be shown validating this new high-density qNPA microarray assay.
This BioMicro Systems study aimed to examine the performance of the MAUI product line, which includes the MAUI Hybridization System with MAUI Mixer hybridization chambers and all-new MAUI Wash System, using a variety of commercial comparative genomic hybridization, expression, and miRNA microarray platforms. In each case, the MAUI product line’s performance was compared to the microarray manufacturers’ recommended protocols. Target material was prepared from commercially available human nucleic acids using a variety of labeling kits. The data analysis consisted of calculating signal-to-noise ratios by dividing the signals of a variety of probes, including negative control probes, by background noise. Where possible, performance was also determined using the microarray manufacturers’ supplied software. The MAUI instruments and consumables produced improved data in all cases, but the magnitude was platform dependent. In general, the higher the molar concentration of immobilized probe and the lower the molar concentration of labeled target, the larger the sensitivity gains produced by the MAUI product line.
The use of DNA microarrays has become an established method for assessing gene expression on a genome-wide scale. However, the substantial amount of RNA required for gene expression analysis is often the limiting factor for this technology. Many studies involving picogram amounts of RNA, such as those involving laser capture microdissection, needle biopsies, etc., are therefore excluded from this technology. Epicentre’s TargetAmp aRNA amplification kits were tested, side by side with the current recommended standard Affymetrix and Illumina protocols. The results of these experiments demonstrated successful use of picogram amounts of total RNA on the BeadChips (Illumina) and GeneChip (Affymetrix) arrays.
Until recently, DNA microarrays were designed with 3k-biased sense-strand DNA probes because available mRNA amplification methods predominantly yielded labeled target RNA or DNA that was complementary to the 3k-portion of mRNA. However, with increasing scientific evidence of the importance of mRNA splice variants and of non-mRNA transcripts, the need to study gene expression patterns of whole transcripts, including all exons, is growing. Thus, new microarrays have been introduced (e.g., Affymetrix’s GeneChip Gene 1.0 ST) for whole transcript and whole transcriptome analysis. However, target amplification methods have not been available to adequately address the capabilities of these new microarray formats.
Using a novel technology, the new RiboMultiplier sense RNA amplification system developed by EPICENTRE Biotechnologies, permits synthesis of microgram quantities of labeled or unlabeled sense RNA (sRNA) target using mRNA or total RNA from small numbers of cells. Labeled sRNA target can be hybridized directly to Affymetrix’s ST 1.0 microarrays. Alternatively, unlabeled sRNA can be used to synthesize labeled ds cDNA target for use on other microarray platforms (e.g., NimbleGen Systems). The Ribo-Multiplier process is as simple as current standard methods, produces target sRNA or cDNA that is highly representative of the transcripts in the sample, and provides full coverage of either mRNA or the whole transcriptome. Amplification from nanograms of total RNA can be completed within one day, and sRNA target product size and respresentation are maintained during multiple rounds of target amplification, permitting amplification from picogram amounts of total RNA (e.g., from laser capture, microdissection, FACS fluorescent sorting, or needle biopsy) within 2 d. Differential expresssion (DE) data obtained for RiboMultiplier-prepared sRNA from commercially available human brain and universal reference total RNA demonstrated excellent correlation with DE data for the corresponding unamplified RNA on different DNA microarray platforms and with known MAQC TaqMan ratios.
Colloidal Coomassie Blue (CCB) is the most widely used stain for protein detection in 1D or 2D electrophoresis gels. CCB is environmentally safe and reduces the disposal cost for used stain and de-staining solution containing methanol. Although many other fluorescence and colorimetric stains are available, CCB remains the first choice of protein gel stain in many laboratories. In the present study, we compared the protein detection in Coomassie-stained gels by the Odyssey Infra Red scanner, Typhoon Multimode fluorescence scanner, and Imagescanner-II densitometer. 1D gel electrophoresis was performed using pre-cast NuPAGE Novex 4–12% Bis-Tris gradient gels (1.0 mm × 20 well) in the MES [2-(N-morpholino) ethane sulfonic acid] SDS buffer system. Pre-stained and unstained protein standards at various protein concentrations were loaded six times. All gels were developed with CCB stain and imaged by Odyssey IR Scanner, using the fluorescence of Coomassie stain in the near infrared region (700 nm), Typhoon fluorescence scanner using excitation source red laser (633 nm) with emission filter 670 BP 30 and with no emission filter, and Imagescanner-II colorimetric method using visible light scanning. All gel images were converted into a dimension-reduced electropherogram. These electropherograms were analyzed by Correct Integration Software System (CISS)1 to determine the sensitivity and linear range of detection. Our data indicate that infrared scanning provides the most sensitive and cost-effective approach for Coomassie-stained gels.
This research was made possible in part by use of the RI-INBRE Research Core Facility supported by Grant #P20RR16457 from NCRR/NIH.
Recent improvements in MS instrumentation and nano-LC reproducibility make a label-free MS-based quantification approach feasible. This technology has the potential to become a significant complement to current quantification methods, such as label-based MS methods (ICAT, etc.) or 2D-gel quantitation methods. The high-throughput compatibility of a label-free approach allows processing of large numbers of samples, which is required to obtain statistically valid quantifications from typical biological samples.
To show the performance of label-free proteomics, we chose a set-up comprising nano-HPLC (Ultimate 3000, Dionex), ESI-QTOF-MS (micrOTOF-Q, Bruker Daltonics), and MS analysis software, and compared the results with a 2D-DIGE study. The analyzed sample for both studies was the human lung carcinoma cell line A549 before and after TGF-beta induction. As our first result, we found that preparing a protein digestion is a very critical step, introducing systematic variances. Therefore, we tested different digestion protocols, allowing an effective and reproducible protein digestion. After determining the optimum digestion conditions, we performed a series of different LC-MS runs and analyzed them with MS analysis software to determine quantitatively the relative amounts of up- or down-regulated peptides. Such statistically significant regulated peptides were put onto an inclusion list, which triggers an automated LC-MS/MS analysis for their identification. Here, we will present the preliminary results of our label-free approach regarding reproducibility of the instrumental setup, and compare it with the 2D-DIGE study.
We have developed a novel digital technology that can be used for non-enzymatic direct multiplexed measurement of gene expression that is ultra sensitive and has a high level of precision even at very low levels of gene expression. This technology has been developed into a fully automated system, the nCounter Analysis System, which enables researchers to examine or validate larger sets of transcripts with many fewer reactions while removing the risk of bias being introduced during enzymatic steps. In this study we examined the technical performance of the nCounter System and compared it with results generated with microarrays, TaqMan, and SYBR Green Real-Time PCR.
The tyrosine kinase protein, epidermal growth factor receptor (EGFR), is mutated in approximately 15% of non–small cell lung cancers (NSCLC) in the Caucasian population. Many studies have linked the presence of these mutations to response with the tyrosine kinase inhibitors gefitinib and erlotinib. There are many methods of detecting EGFR mutations, including direct DNA sequencing. However, since the majority of NSCLC cases will be wild type with respect to EGFR, the ideal mutation testing method would be a scanning technology that will allow for the rapid elimination of all wild-type cases. Here, we describe a method of using unlabeled oligonucleotide probes and high-resolution melting analysis (HRMA) to detect the most common EGFR mutations.
Primers and unlabeled probes were designed to amplify and span the most common mutated sites in EGFR exon 19 and exon 21. LC Green plus, a double-stranded DNA dye specific for high-resolution melting, was added to the standard PCR reagents before amplification. After PCR, the samples were melted at 0.3°C/sec using the HR-1 (high-resolution melting) instrument.
The derivative melting curve of the probe-target duplexes was characteristic of the genotype spanned by the probe. DNA sequencing was used to confirm abnormal results. NSCLC samples containing EGFR mutations L858R, del 746–750, or del 747–752 P753S were detected with the use of unlabeled probes. The use of unlabeled probes improves the detection of mutations in which the tumor is homozygous or mutant allele increased with subsequent dilution of the normal.
The most common EGFR activating mutations can be rapidly and inexpensively screened for with the use of unlabeled probes and HRMA. The presence of EGFR activating mutations in non–small cell lung cancer could help identify which patients may benefit from EGFR kinase inhibitors.
With remarkable advances in genomic technologies, the National Cancer Institute established the Core Genotyping Facility (CGF) to investigate the contribution of germline genetic variation to cancer susceptibility and outcomes. Working in concert with epidemiologists, biostatisticians, and basic research scientists in the intramural research program, the CGF has developed the capacity to conduct genome-wide association studies and candidate gene approaches to identify the heritable determinants of various forms of cancer.
Utilizing technologies by Illumina, Affymetrix, Applied BioSystems, and Fluidigm, the CGF has created a laboratory facility capable of high-throughput genotyping and detailed scientific follow-up studies. To support these efforts, the CGF has its own analytical and programming group, whose efforts are focused on the development of new tools for data handling and analysis. In addition to a process-driven LIMS system, the CGF is designing a project management interface to assist in tracking the many sample-handling and genotyping processes that pass through its doors.
In the current environment, the CGF is capable of taking collected samples from the epidemiologist and following a circular path of genetic discovery, analysis, reproduction, and reporting using the laboratory and analytic tools contained within the organization. This allows rapid response to large-scale discoveries. As the climate of genetic research changes, the CGF has entered into collaboration to explore next-generation sequencing technology by Roche 454 and is looking forward to other new technologies.
This project has been funded in whole or part with federal funds from the NCI, NIH, under contract N01-CO-12400.
It has been estimated that by the year 2030, the number of individuals age 65 and older will reach 70.3 million, constituting approximately 20% of the US population. As immune function is known to decline with age, there will be a rapid growth in the population of individuals with both increased disease susceptibility and higher rates of morbidity and mortality due to infectious disease. The capacity to generate protective T-cell responses against newly encountered pathogens is dependent on the maintenance of a diverse naïve T-cell repertoire, and the size of the naïve T-cell repertoire is thought to decline with aging. In this study, we used the well-characterized mouse model of influenza A virus infection to investigate the impact of naturally occurring, age-related changes in the naïve T-cell repertoire on the capacity of aged mice to mount effective CD8 T-cell responses against a primary influenza virus infection. Using MHC Class I tetramer staining reagents generated by the Institute’s Molecular Biology Core Facility (MBCF), it was found that the majority of aged mice had impaired CD8 T-cell responses to the known immunodominant viral NP/Db epitope. Through the use of the MBCF spectratyping services, it was further established that the diversity of TCR Vβ chain usage by NP/Db-specific CD8 T-cells present in most infected aged mice was quite limited. These findings, obtained with the vital assistance of the MBCF, may be relevant for the design of vaccines and/or therapies aimed at reducing the risk of infectious disease within elderly populations.
Hematopoietic stem-cell transplantation (HSCT) involving multiple donors (MD) is an increasingly common occurrence because of graft failure, or associated engraftment complications. As in all HSCTs, monitoring donor-recipient chimerism (CHM) quantitatively is a critical means of assessing graft status. However, MD chimeras are often complicated by extensive allelic sharing and chimeric inequalities between donors, so that current quantitative analytic approaches needed to be modified and extended to accommodate complex chimeric states. This study was undertaken to develop an analytic algorithm that is based on the true quantitative nature of the STR data when the reliability of the measurements has been validated. Our approach was to evaluate the stages in CHM analysis in terms of the impact of the possible allelic configurations in MD chimerism and the numerical relationships of alleles when shared. The four principle stages are: (1) identifying informative loci; (2) assessing reliability of the measurements;1 (3) resolving the components of shared alleles in informative loci with reliable measurements; (4) computing chimeric level based on the foregoing stages, and an expanded version of the standard formula for donor %CHM. From these considerations, we have been able to develop guidelines for each stage that can serve as an algorithm for the analysis of complex chimerisms of several forms, thereby enabling routine CHM monitoring in such cases. The algorithm is flexible, so that its stages could be used in concert, or independently to accommodate a range of post-transplantation monitoring requirements, including multi-donor situations that are sequential or simultaneous, iatrogenic or in utero.
Smoking is an established risk factor for periodontitis and is responsible for 42% of periodontitis in the United States. Both bacterial plaque and smoking play important roles in periodontitis, but their interaction is not well understood. The objective of this initial study is to determine how similar the bacterial populations are between current smokers and those who were former smokers—i.e., if a person quits, does the bacterial population change? The nature of the bacterium community was characterized by terminal restriction fragment length polymorphism (TRFLP) analysis. TRFLP analysis measures population diversity by observing differences in genomic DNA sequences as indicated by the differing location of restriction endonuclease sites in a conserved region of the genome. Two oligonucleotides labeled with FAM or VIC were used to amplify a conserved region of the 16s rRNA gene from total subgingival DNA, and were subsequently digested with either MspI or HhaI. Two methods of sample cleanup were compared: (1) dialysis and (2) solid-phase reversible immobilization, which was necessary to remove the digestion buffers prior to analysis on an automated fluorescent capillary DNA analyzer. The two methods were found to be comparable. The fragment patterns were found to be significantly different after statistical analysis with the Bray Curtis Similarity Index, so cessation of smoking does alter the subgingival bacterium population in cases of periodontitis.
Accurate mutation calling and quality data have been identified as key components of direct sequencing by many clinical researchers. We used Variant Reporter software to detect mutations in a large volume of datasets. This analysis tool integrates improved algorithms for SNP detection that are trained to discover accurate sequence variations and report review status for traceability. It helps to create expressive Quality Control Data reports for large datasets and annotated projects that contain trace files and data annotation. Data-sharing abilities between users, such as between core facilities and end users, will be demonstrated. The guided workflow gives a new or advanced user confidence in a short period of time. In this poster we will highlight how core facilities can use the new Quality Control metrics and how end users can share the accurate results.
Deletions and duplications in genomic DNA have been implicated as pathogenic mutations in many diseases. Traditionally, detection of these types of mutations is done using Southern blot hybridization or fluorescence in situ hybridization, techniques that can be laborious and time-consuming, and require high quantities of starting material. In this study, we present analysis of a semi-quantitative multiplex PCR-based method that uses relative quantitation of fluorescently labeled fragments. Fragments from BRCA1, BRCA2, 9p21, and MMR (MSH2) regions were tested using labeled probes from DNA that had been isolated from blood. Amplified samples were then run on an Applied Biosystems capillary electrophoresis platform, and the data were analyzed in GeneMapper software v4.0. After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper software v4.0 report manager feature and verified using the dye scale functionality. Our results will highlight an easy-to-use method whose optimal workflow and analysis can be used for both small- and large-scale studies.
DNA variant detection in resequencing offers key information for medical research and clinical applications. We have developed Variant Reporter Software v1.0, which includes improved algorithms for the automated detection and scoring of sequence variants in an amplicon-based workflow. In this poster, we provide results of validation experiments performed on large, annotated re-sequencing datasets in order to demonstrate the accuracy and sensitivity performance of the algorithms. For mutation and SNP detection, the Variant Reporter algorithms reduce error rates by an order of magnitude when compared to competing software solutions.
Integrated with the KB Basecaller v1.4, Variant Reporter Software takes advantage of mixed base-calling information computed during the primary analysis. The downstream algorithms can analyze up to 5000 samples within 5 min and detect heterozygous mutations with high accuracy under both single-specimen and multi-specimen scenarios. The algorithms assign a confidence score to each point variant prediction, and phred-style quality values to each specimen consensus call. In conjuction with the automated scoring, the Variant Reporter Software offers novel UI features to facilitate filtering and reviewing the variant predictions. The software thus allows users to tune the finishing process according to their application-specific requirements and cuts substantially the manual review burden needed to reduce a Sanger-based resequencing methodology to a standard and reliable practice.
The demand for high-purity peptides is increasing. Recent successes of peptide drugs, including enfuvirtide, eptifibatide, and teriparatide, have renewed interest in this compound class from pharmaceutical companies. Small synthetic peptides to large cellular produced peptides are being investigated for possible therapeutic benefits. Both can be difficult to purify to high levels, >98%, because of the very similar products, many times differing by only one amino acid. Optimized purification techniques are required to meet these high purity demands in an economical manner. Reversed-phase chromatography, because of its high resolving power, has been the technique of choice for achieving the high level of purity necessary in the pharmaceutical industry. Utilizing a new reversed-phase silica, we show how improved selectivity and resolution between the target peptides and impurities is increased as well as how unique bonding chemistry and silica surface treatment can increase recovery and loading capacity.
To enhance chromatographic resolution, high-performance liquid chromatography (HPLC) has followed a distinct evolution toward smaller-diameter packing materials. We herein investigate and compare the use of nanobore columns (75 μm) possessing integrally fritted tips packed with conventional (5 μm diameter) and smaller (1.7 μm diameter) silica-based C18 media. The packed columns demonstrated full compatibility in peptide analysis using a sub-2-μm reversed-phase media. The operating pressure (ca 3000 psi at 300 nL/min) of a 75 μm × 10 cm, 1.7 μm C18 packed-tip column is within the limits of conventional HPLC. Improved chromatographic performance (decreased chromatographic peak width) yielded a typical twofold increase in ion intensity compared to conventional 5-μm media, with an attendant increase in MS signal-to-noise ratio. Selected ion peak widths (FWHM) of less than 4 sec (20 min gradient to 50% B) were typical with the 1.7-μm media, yielding a calculated peak capacity twofold higher than conventional 5-μm media. Further improvement with the 1.7-μm media may be possible at higher flow rates. The loading capacity of the 1.7-μm media appears to fall off at approx. 100 fmol/peptide; more study of loading capacity is required. Retention-time reproducibility for replicate injections of approximately 2 sec is possible, although careful attention to column conditioning between injection cycles is warranted. Analysis time reduction and/or improved peak capacity with longer columns should be possible with higher pressure LC pumps (ca 10,000 psi), and is under further investigation.
Protein purification plays an essential role in the development of new drug therapies. By generating and studying multiple variations of a single target protein in a systematic approach, we are able to gain insight necessary to better develop new drug compounds. Therefore, achieving higher throughput for protein purification will greatly help to move drug discovery efforts forward.
In order to increase our protein purification throughput, we have implemented two purification protocols utilizing the AKTAxpress liquid chromatography instrument from GE Healthcare for purification of polyhistidine-tagged proteins. The first standard protocol involves two column purification steps: immobilized metal affinity chromatography (IMAC) followed by size exclusion. High protein purity is achieved by washing the target protein from an IMAC column after on-column removal of the tag by protease digestion. Any uncleaved target protein and most nonspecifically bound contaminants remain bound to the column, thereby amplifying the specificity of the IMAC. This entire protocol is automated into a single program controlling four AKTAxpress units, allowing for the complete purification of four separate proteins in parallel. For noncleavable polyhistidine-tagged recombinant proteins, we have developed a second protocol encompassing four column purification steps divided into two program runs. The first program utilizes an IMAC column followed by desalting and ion-exchange columns. In order to overcome the problem of extreme dilution of low expressed proteins during the ion-exchange step, fractions are then pooled and concentrated off-line before loading onto a size-exclusion column as a second program run.
As a result, we have purified up to four different protein constructs to greater than 95% purity in less than 3 d. Final protein yields have ranged from 0.5 mg to 14 mg from 2–4 L cell cultures. The implementation of these two protocols on the AKTAxpress has increased our large scale protein purification throughput by fourfold.
The liquid chromatography front end of a mass spectrometer is a critical aspect of modern LC/MS/MS systems. Most proteomics analyses and metabolomics analyses require nanoliter flow rates for optimal sensitivity. While many high-flow systems are run using split flow to get down to nanoliter flow rates, the recent trend is to use splitless flow systems capable of pumping at nanoliter flow rates. We have taken such a system, containing a full-service autosampler, requiring no external gasses, pre-configured and ready optimized for proteomics studies (shortest flow lines and lowest dead-volumes) and used it with an Orbitrap XL mass spectrometer. We analyzed the capability of the system for quantitative applications of cellular signaling phosphorylation events using peak area ratio analysis between different stimulated cellular conditions. The reproducibility and resolution of the system was sufficient for these analyses since resolution for peptides was ~8–9 sec peak width at half height and the reproducibility for tryptic peptides ranged from 0.22 to 0.34 %RSD. We also show an example where peak area ratios were used to quantify the ratio of mutated peptide to wild-type peptide under different protein interaction conditions from cancer cells. The system was further used to sequence peptides from 80-million-year-old fossil bone with unparalleled sensitivity from the combination of the nanoflow HPLC and the Orbitrap, representing the oldest sequences recorded to date.
Reversed-phase nanobore HPLC combined with electrospray ionization tandem mass spectrometry is widely used for identifying proteins in biological samples. With maximum robustness as a primary objective, sample traps provide multiple advantages. By effectively desalting and concentrating samples online, sample traps improve analytical column longevity and sample throughput. Here, we have evaluated a novel nano-LC ESI-MS sample trap possessing an optically clear, colorless insert; this insert contains reversed-phase resin and a chemically inert fluoropolymer sealing mechanism. Using a commercially available peptide standard mixture, the performance of this sample trap configuration was assessed by evaluating its loading capacity, operating back pressure, and peak shape. The efficacy of the optically clear sample trap was compared to commercially available sample traps and its superior benefits to nano-LC ESI-MS are demonstrated.
Due to the large number of proteins and wide range of relative abundances in complex proteome samples (i.e., whole cell lysates), a significant amount of resolution is required prior to MS analysis in order to identify, characterize, and/or quantitate low-abundance proteins. Proteomics researchers currently use a wide variety of separation protocols to separate proteins and/or peptides prior to MS analysis, including 1D or 2D gels, 1D or 2D protein LC, and 1D or 2D peptide LC, but compromises often need to be made to minimize sample losses and total analysis times. This study introduces a new automated 3D peptide separation protocol that offers maximum peptide resolution and recovery while minimizing total MS analysis time. Using a proteomics sample prep workstation coupled with a biocompatible MDLC, 500 μg of an E. coli lysate tryptic digest was separated by 1D (low-pH RP), 2D (SCX-low-pH RP), or 3D (high-pH RP-SCX–low-pH RP) prior to MS/MS analysis. The 3D protocol has the advantage of allowing online concentration and desalting of digests (necessary prior to SCX) prior to offline fractionation using high-pH RPLC. Fractions from the first dimension (pH adjusted to 3) were loaded on the second-dimension SCX column, which removes detergents and excess acetonitrile from samples, prior to online salt step fractionation. Since the 3D protocol produces more total fractions, a high-throughput method is required for the LC/MS runs in order to provide a comparable total analysis time. Using a new “Plug and Play” nano/capspray source that provides high sensitivity over a wide flow range, we were able to do an optimized comparison of the 1D, 2D, and 3D protocols to determine the relative advantages and disadvantages of these three different separation schemes.
In proteomic studies, two-dimensional liquid chromatography (2D-LC), using a strong cation exchange (SCX) separation followed by gradient elution on a reversed-phase (RP) column is frequently applied prior to MS analysis to facilitate analysis of complex samples. Most 2D LC systems require sophisticated ternary or quaternary gradient generation setups to be able to deliver the salt elution steps from the ion exchanger onto the RP column, and accurate gradient elution to elute the peptides into the MS. Here, we describe a simple, automated SCX/RP 2D separation strategy achieved using a split-free, nanoscale LC system already optimized for use within an LC-MS setup.
To evaluate system performance, we analyzed the proteome of mouse placental cells. The cytosolic fraction was subjected to cysteine reduction and alkylation with iodo-acetamide followed by trypsin digestion. The lysate was purified and analyzed by 1D and 2D methods on a split-free nanoscale LC system (EASY n-LC Proxeon, Odense, Denmark) coupled to LTQ-Orbitrap (Thermo-Fisher, Bremen, Germany). The 2D separation was performed using a 10-step salt elution varying from 0.05 to 0.5 M ammonium acetate.
The auto-sampler component of the EASY-nLC and standard injection programs were used to deliver the salt elution steps, thereby transforming a 1D LC system into a 2D system. The viability of this method was demonstrated by comparing LC-MS data obtained following 1D and 2D separations. The 1D analysis yielded about 600 protein identifications, whereas 2D analysis identified approximately 2000 proteins. These results clearly demonstrate the significant improvement that can be achieved by slight modifications of the 1D nanoscale LC system. Far more information was obtained with only minimal modification of the LC component in the LC-MS analysis.
In today’s high-performance liquid chromatography (HPLC) systems, common detectors typically measure ultraviolet light absorption or refractive index changes. Recently, a new stand-alone absolute size exclusion chromatography (ASEC) detector was developed. Here, dynamic light scattering (DLS) is used to analyze intensity fluctuations in the light scattered from a laser beam in an online flow cell. DLS provides a direct measure of the protein size, as opposed to the SEC-derived size extracted from a calibration curve. As different peaks elute, the diffusion coefficient and size of the molecules is determined in real time without column calibration or knowledge of any sample-specific constants. The ASEC method can quickly identify peaks and quantify presence of aggregates or oligomeric species. In addition, size versus mass relationships for globular proteins are well known, and can be used to calculate the molecular mass from the DLS-measured hydrodynamic size.
Data for antibodies, growth hormone, carbonic anhydrase, and lysozyme are presented. The technique is reliable and simple, yet provides additional information to complement other biophysical characterization approaches.
Nanoflow liquid chromatography coupled with nanoelectrospray (nano-LC-MS) has proven to be an invaluable tool for sensitive peptide and protein analysis. As the demand for more complex proteomic analyses grows, instrumentation and experimental methods that support higher-resolution peptide separations are required. We present new hardware developments and columns for improving the resolution of nano-LC separations while maintaining the benefits of reproducible separations and high mass sensitivity.
We have developed a split-less nano-LC system that addresses three of the main demands for high-resolution peptide analysis—very low gradient delay volumes, increased range of flow rates, and operating pressures up to 10,000 psi.
The system builds on the direct pumping, feedback controlled fluid delivery developed and described previously for conducting reproducible chromatography at flow rates of 100–1000 nL/min. Seventy-five-micron I.D. columns with lengths up to 30 cm and packed with sub-3-micron particles were used for increasing resolution of peptide separations. The effects of column length, particle size, temperature, and gradient slope on the separation of peptides in complex protein digests will be demonstrated.
Solid phase extraction (SPE) is a simple and widely used technique for desalting and concentrating peptide and protein samples prior to mass spectrometry analysis. Often, SPE sample preparation is done manually and the samples eluted, dried, and reconstituted into 96-well titer plates for subsequent LC MS/MS analysis. To reduce the number of sample-handling stages and increase throughput, we developed a robotic system to interface off-line SPE to LC-ESI-MS/MS.
Samples were manually loaded onto disposable SPE tips that subsequently were connected in-line with a capillary reversed-phase (RP) column. Peptides were recovered from the SPE precolumn and separated on the RP column using isocratic elution conditions, and analyzed by electrospray tandem mass spectrometry. Flow was delivered by two nanoflow piston pumps operated with Advanced Flow Control (AFC) (Proxeon, Odense, Denmark). Using a modified autosampler for mounting and disposal of the SPE tips, the SPE-LC-MS/MS system could analyze eight samples per hour, and up to 96 SPE tips in one batch.
The chromatographic performance of the SPE-LC system was evaluated in terms of peptide ion peak widths, column peak capacity, and retention time reproducibility based upon the analysis of tryptic BSA and a 12 protein component mixture. Peptide mixtures eluted within approximately 5 min, with individual peptide peak width of ~5 sec (FWHM), making the SPE-LC suited for high-throughput analysis.
The relatively high sample throughput, sufficient separation power, and high sensitivity makes the automated SPE-LC MS/MS setup attractive for proteomics experiments, as demonstrated by the identification of the components of simple protein mixtures and of proteins recovered from SDS-PAGE and 2DE gels.
The analysis of proteins and peptides by reversed-phase high-performance liquid chromatography (HPLC) is important in the development of well-characterized biotechnology pharmaceuticals. Since polypeptides interact only slightly with the stationary phase after desorption, it is known that short columns are suited for fast separations. By decreasing the particle size of HPLC packings, column efficiency is increased. We demonstrate the use of 1-cm length HPLC columns packed with 1.5-μm, 500A, C18-bonded silica at high flow rates, with moderate backpressures (<3000 psi), using conventional HPLC systems. A linear velocity of 1200 cm/h, four times higher than typical 5-μm columns, allows for ultra-fast, 1-min separations of proteins with broad physicochemical characteristics. Closely related insulin variants (bovine, sheep, human) may be separated in under 1.5 min; this represents a 15-fold decrease in analysis time as compared to separations with traditional HPLC columns and media. While the typical HPLC run time for the separation of hemoglobin chains on conventional columns ranges from 25 to 60 min, runs under 2 min may be realized with the large-pore, sub-2-μm silica. Accordingly, five different hemoglobin samples (from different animal species) may be compared after a total of only 14 min. Furthermore, intact, high-molecular-weight IgG antibodies may be analyzed in less than 4 min. The large-pore, sub-2-μm HPLC columns represent a new technology for the characterization of biopharmaceuticals in an era when increasing sample throughput has become a high priority in most laboratories.
WikiLIMS is a suite of programs for managing laboratory data. It uses MediaWiki (the software behind Wikipedia) to store structured information in a semantic web. Any standard Web browser is sufficient to read and write the wiki-based experimental metadata. Larger laboratory data files are stored in a Web-accessible RAID file system. Control panels within the wiki allow users to launch and monitor site-specific analyses via Sun Grid Engine. The MediaWiki engine allows users to easily annotate records, and administrators to monitor changes in the data. The system deployed at the Naval Medical Research Center currently contains 5000+ records of samples, Roche/454 GS-20, and GS-FLX sequencer runs, and Nimblegen and Afffymetrix microarray experiments.
In today’s proteomics research, it is necessary to use different techniques, instrumentation, and bioinformatics tools to manage the large amount of heterogeneous data with an automatic quality control to produce reliable and comparable results. Therefore, a data warehousing system including a data-processing pipeline is mandatory for data validation and comparison. The proteome bioinformatics platform ProteinScape has been proven to cover these needs.
For result enhancement, ProteinScape features different tools for MS data processing, such as automatic searching of large MS/MS datasets with several database search engines, automatic calibration and contaminant filtering of PMF data, and protein list generation from MS/MS results of complex protein mixtures. To generalize the reprocessing of diverse datasets, a guideline (forum.hbpp.org) has been set up defining the workflow of protein identification, which is fully implemented within ProteinScape. Peptide identifications from different search engines were analyzed by the ProteinExtractor algorithm to reduce the list by the false-positive hits using a decoy sequence database strategy. In a consecutive step, the algorithm compiles a number of peptide lists from different origins (e.g., different workflows, different search engines, repetitive experiments, different MS-based techniques (MALDI-TOF, ESI-IT), and any combination) into a single consensus protein list providing a final protein list with a known false-discovery rate.
An export tool allows us to transfer all relevant HUPO BPP data from ProteinScape into PRIDE. An EU-funded project, ProDaC, will coordinate systematic data collection in public standards-compliant repositories like PRIDE. This will cover all aspects, from generating MS data in each laboratory to assembling all the annotation information and storing it together with identifications in a standardized format. This private dataset at PRIDE can be used for reviewing processes of all journals. After publication, the private data can be made public to be used as knowledge of the whole scientific community.
Nanodiscs are self-assembled soluble discoidal phospholipid bilayers encirculated by an amphipatic protein (membrane scaffold protein, MSP) that provide a functional stabilized membrane disc for the incorporation of membrane-bound and membrane-associated molecules. Integral membrane proteins in nanodiscs adopt a uniform environment that resembles biological membranes, and the lipid composition in the immediate surroundings of the protein can be controlled. Additionally, its oligomerization state can be varied by careful control of protein:MPS stoichometry and by selection of MSP with a suitable length that leads to a disc area that can accommodate the desired oligomerization state. The scope of the present work is to investigate how nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs.
Cholera toxin and its glycolipid receptor GM1 constitute a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodisc containing the glycolipid receptor GM1 in lipid bilayers, enabling measurements of binding of its soluble interaction partner cholera toxin B subunit to the receptor with the sensorchip-based surface plasmon resonance (SPR) technology. The measured stoichiometric and kinetic values of the interaction are in agreement with those reported by previous studies, providing evidence that nanodiscs can be employed for kinetic SPR studies.
In addition to immobilization on sensorchips, we have used FLAG-tagged GM1 nanodiscs for co-immunoprecipitation with FLAG antibody–coated agarose beads from enterotoxigenic E. coli lysates. The co-immunoprecipitated protein was identified by mass spectrometry as heat-labile toxin, a homolog of cholera toxin.
Melittin is the principal active component of bee venom. Melittin is a peptide consisting of 26 amino acids and kills bacteria by interacting with the bacteria’s membrane. Production of melittin is stimulated upon bacterial injury as a defense mechanism. The melittin peptide is known to spontaneously induce transmembrane pores. It is believed that bundles of the lipid-spanning peptides (melittin molecules) collectively form a pore through which hydrophilic molecules can diffuse across the lipid bilayer.
Layerlab provides a technique for anchoring liposomes to virtually any chemically modifiable surface through the hybridization of complementary DNA strands. Layerlab has developed a commercially available kit, which facilitates handling and immobilization of membrane-bound or membrane-associated biomolecules to surfaces, and which has been designed for the surface plasmon resonance (SPR)-based Biacore instruments and quartz crystal microbalance (QCM-D)-based Q-Sense instruments.
The QCM-D technique has the advantage that it gives structural information on the adsorbed biomolecules. The structural information is obtained by monitoring the visco-elastic properties of the adsorbed film of biomolecules. The action of melittin influences the structure of the liposome membrane, which makes the QCM-D technique an excellent choice to measure these types of events.
Here we show results from measurements on the action of melittin on the lipid membrane of lipsosomes, immobilized using the Layerlab technology, on sensor surfaces of the Biacore and the Q-Sense instruments.
The Vincent Coates Foundation Mass Spectrometry Laboratory is named in honor of a generous gift from Vincent and Stella Coates, given to support the mass spec & proteomics facility as a shared core resource. It is a Bio-X core facility, embodying the Bio-X spirit of interdisciplinary communication and collaboration. It is also the proteomics core facility of the Stanford Comprehensive Cancer Center. The laboratory’s expertise and support are available to researchers throughout Stanford University & Medical Center and worldwide.
Beyond making available state-of-the-art, user-friendly facilities and services, the laboratory enables education, methods development, and new applications development, designed to meet the rapidly evolving needs of researchers. Due to the essential information that mass spectrometry provides to researchers in the fields of the physical and life sciences, medicine, and engineering, the laboratory serves as an “intellectual watering hole” at the crossroads of diverse disciplines.
Routine mass spec and proteomic services include molecular-weight determination, MSn, LC-MS, high-resolution MS, protein identification by proteolytic digest, nano-LC-MS/MS and database search, standard and long-column capillary chromatography and MudPIT, and more. Consultations are essential for custom work such as complex proteomic analysis, quantitation (e.g., metabolic or environmental studies), de novo peptide sequencing, new application development, and other projects.
Contact information and more about the lab can be found at http://mass-spec.stanford.edu.
The Mass Spectrometry and Proteomics Resource of the Yale University W.M. Keck Biotechnology Resource Laboratory provides a very broad array of protein and mass spectrometry analyses. These range from classical protein analyses such as amino acid analysis and Edman sequencing, to FT-ICR-based peptide/protein disease biomarker discovery and analysis of protein post-translational modifications. Focus is placed on protein profiling approaches, including iTRAQ, ICAT, differential (fluorescence) gel electrophoresis (DIGE), SILAC, MudPIT, serum/plasma biomarkers analysis, phosphoproteome analysis, and 2D-LC comparisons. Routine MS analyses include protein identification from enzymatic digests (LC-MS/MS and MALDI-TOF/TOF), analysis of small molecules, intact proteins, oligonucleotides, and exact mass (<1 ppm) determination. The FT-ICR mass spectrometer (9.4T Apex-Qe FT-ICR) offers additional methods of fragmenting peptides and proteins that are not available on Q-TOF type instruments, which makes it a unique platform for locating sites of modification (e.g., glycosylation and ubiquitinylation). Two newer technologies are phosphoproteome analysis and translational proteomics. The phosphoproteome analysis includes an optimized TiO2 enrichment, identification of sites of phosphorylation, and relative quantitation of phosphorylation changes. The translational proteomics is based on quantitative mass spectrometry analysis of the in vivo concentrations of multiple, pre-selected biomarker proteins. Lastly, to handle the large amounts of data, particularly from the high-throughput proteomic technologies, we have developed an open-source bioinformatics tool called the Yale Protein Expression Database (YPED). YPED is used for storage, retrieval, and intergrated analysis of these large datasets with a link to the PANTHER Classification System to aid in biological interpretation. Examples will be given of several of the services offered.
Using isotope ratio mass spectrometry (Thermo Delta Advantage with H-device), the Center for Nutrition Research Unit (CNRU) Mass Spectrometry Core Facility provides an array of metabolic analyses, including measurements of total energy expenditure (TEE) using doubly labeled water (DLW), total body water (TBW) using deuterium oxide (D2O), and metabolic measurements of 13C in breath CO2. TEE and TBW for body composition or body water turnover are measured using urine or plasma samples. Global metabolism of macronutrients (e.g., 13C-labeled monosaccharides, fatty acids, or amino acids) can be measured using breath CO2. Our well-equipped facility also includes a GC/MS, three triple-quadrupole mass spectrometers, two ion traps, and an ESI-TOFMS. The diverse technologies available allow investigators to obtain orthogonal, confirmatory data.
The CNRU Mass Spectrometry Facility not only provides analytical services and expertise to investigators and collaborators affiliated with the Human Nutrition Center, but also works with other groups within the University of Colorado Denver, including the Schools of Medicine and Pharmacy, graduate students and researchers at our Downtown Denver Campus, University Hospital, and Children’s Hospital. We also offer our services to outside and industrial partners. In addition to service work, our scientists engage in both their own and collaborative research.
The Proteomics and Mass Spectrometry Facility at the Donald Danforth Plant Science Center (http://www.danforthcenter.org/msb/) is equipped with state-of-the-art technologies for the detailed study of a wide range of biomolecules. The facility provides both full- and self-service capabilities to both internal and external clients at competitive rates. The facility offers fast, high-quality, specialized analytical services including: protein extractions, liquid chromatographic separations; high-resolution 1D/2D gel electrophoresis; gel image analysis and protein expression analysis; high-throughput protein spot excision; in-solution and in-gel protein digestion; high-throughput protein identification; accurate protein molecular-weight analysis; protein covalent/non-covalent complex analysis; biomolecule interactions (surface plasmon resonance); small molecule separation/structure determination; protein post-translational modification analysis, and peptide de novo sequencing. Major instrumentation includes: QSTAR XL Q-TOF MS/MS system (Applied Biosystems), 4000QTRAP LC-MS/MS system (Applied Biosystems), Voyager-DE STR MALDI-TOF MS (Applied Biosystems), GCQ GC-MS (ThermoFinnigan), nanoflow HPLCs (LC Packings/Eksigent), System Gold HPLC (Beckman Coulter), Shimadzu HPLC system (Shimadzu), Ultra Performance LC (UPLC) (Waters, Inc.), Biacore2000 (Biacore, Inc.), 1D and high-resolution 2D gel electrophoresis systems (BioRad and Amersham Biosciences), Typhoon 9410 variable mode imager (Amersham Biosciences), GelPix high-throughput protein spot excision system (Genetix, Inc.), and a MultiProbe II automatic protein digestion and handling system (Perkin-Elmer, Inc.).
Protein intact mass, identification, and characterization are a few of the many applications that the facility performs. For proteomics applications, the Q-TOF and MALDI-TOF instruments are well suited for analyzing both small peptides and large proteins. The QTOF instrument can perform exact mass measurements for molecular formula determination and can be coupled with online separations using nano-LC for de novo sequencing and modification analysis. The 4000QTRAP system serves as a powerful instrument for metabolomic profiling, label-free peptide quantitation, and advanced post-translational modification analysis, and our Waters UPLC system is set up to perform both free and hydrolyzed amino acid analyses.
Aging is a complex process affecting a wide variety of functions, including the development and maintenance of the immune system. Loss of thymic function in aging leads to an attenuated immune system and increased morbidity in aged individuals. The objective of this study was to define and characterize whole-tissue proteomes of pediatric versus geriatric human thymus, and identify thymic proteins that are associated with robust thymopoiesis in young thymus and diminish with age.
We have utilized gel electrophoresis followed by MALDI-MS/MS to generate proteomic profiles of young and old thymus. Spatial distribution and compartmentalization of thymic proteomes were analyzed utilizing laser capture microdissection (LCM). Captured cortical and medullary regions were subjected to direct MALDI profiling. We also have incorporated MALDI imaging MS (MALDI-IMS) to further document spatial distribution of thymic proteins in young thymus tissue.
Proteomic analysis revealed evidence for differential expression of proteins between pediatric and geriatric thymus tissue. We identified proteins with functions that include structural, stress resistance, enzymes, cytokines, and adhesion in young thymus that were absent in aged thymus. Comparison of proteins in thymic cortex (immature thymocytes) versus thymic medulla (mature thymocytes) profiles showed unique LCM proteomic profiles in each of the thymic compartments. Finally, we employed MALDI-IMS to establish the spatial distribution of selected protein masses using snap-frozen pediatric thymus tissue sections. Both abundant proteins with scattered distributions and less abundant proteins with more localized distributions were mapped. Overlays of different protein masses provided additional information on the spatial distributions of multiple analytes.
Further studies utilizing the proteomic approaches above will enable us to monitor the expression, post-translational modifications, and spatial distribution of thymic protein protective factors during aging.
Monoclonal antibodies (MAb) have been used as therapeutic agents for the treatment of many diseases. Mutations in the complementary domain region (CDR) and the Fc region have several impacts on the antigen specificity as well as stability of the MAb. Therefore, it is necessary to monitor such mutations in order to maintain MAb activity and selectivity. We previously described a method for the de novo sequencing of MAb using a combination of on-PVDF membrane digestions, Edman degradation, and LC-MS/MS. The bottleneck of this technique was mainly the digestion steps as well as the informatic analysis. Here we explore an alternative digestion method performed on nitrocellulose (NC) membrane.1
In this approach, the heavy chain (HC) and light chain (LC) of a MAb were separated on SDS-PAGE under reduced conditions. The protein was then electroblotted onto NC membrane. Bands corresponding to the HC and LC were excised, and unoccupied sites on the membrane were blocked with PVP-360 (0.5% in 0.1 M acetic acid). Protein was digested with various proteases, such as trypsin, chymotrypsin, Asp-N, pepsin at 37°C for 1–4 h in order to obtain overlapping peptides. The NC membrane was then dissolved in a mixture containing 70:30:1 acetonitrile:methanol:TFA. The digest mixture was diluted with 0.1% TFA such that final concentration of acetonitrile was ~25% in order to precipitate NC membrane. The supernatant was spun down and the mixture was subjected to LTQ-MS-MS/MS for sequencing information.
Using this digestion method, higher sequence coverage was obtained in a short time, which allowed us to rapidly produce peptides for the de novo sequencing of an entire therapeutic MAb.
Three common alleles and many rare variants of vitamin D binding protein (DBP) exist. The protein is also known to be at least partially O-glycosylated. According to the literature, however, several issues remain ambiguous regarding its phenotypic expression: (1) The extent to which the various allelic forms are glycosylated; (2) the degree of glycosidic homogeneity and possible existence of N-glycosylation; (3) the precise site(s) of glycosylation event(s); and (4) the statistical association, if any, that one particular allelic form, GC*1S, has with type 2 diabetes.
A study of over 100 individual plasma samples using affinity capture followed by analysis of the intact protein by LC-ESI-TOF was undertaken to resolve these issues.
Findings demonstrate that all DBP allele products except GC*2 are modified (10–25 mole%) with a (NeuAc)1(Gal)1(GalNAc)1 trisaccharide and, to a much lesser extent (1–5 mole%) with a (Gal)1(GalNAc)1 dissaccharide. GC*2 protein contains the disaccharide but remains completely free of the trisaccharide, even in heterozygous individuals possessing a second gene product that is modified with the trisaccharide. Previously, the (Gal)1(GalNAc)1 dissaccharide had only been inferred to exist via DBP activity studies, and only suspected as a modification of the GC*2 protein. Thus, all allelic forms of DBP except GC*2 appear to possess two O-glycosylation sites occupied by separate, yet consistently isomass oligosaccharides and, despite a consensus sequence, lack N-glycosylation. Data also provide strong evidence for the site of trisaccharide glycosylation. Finally, in addition to documented glycation of diabetic DBP (separate from glycosylation), a positive correlation was found between possession of the GC*1S allele and development of type 2 diabetes.
The manner in which these discoveries were made, coupled with the near impossibility of making these findings using alternative proteomic approaches, suggests that targeted analysis of intact proteins across populations is critical to the investigation and ultimate understanding of human proteins.
Circulation of blood throughout the body may allow changes in overall health of an organism to be reflected in the abundance of proteins and peptides in serum. Due to serum’s complexity, pre-analytical sample simplification and separation are needed prior to MS proteomic analysis. Use of a reversed-phase capillary liquid chromatography column (cLC) coupled to a mass spectrometer allows for online separation and analysis of complex samples, like serum, as part of biomarker discovery. Even after sample simplification via acetonitrile precipitation, data files for a single sample were ~1 gigabyte. The size and complexity of serum datasets exceeded the capacity of currently available software packages to adequately analyze data. Hence, we adopted a manual approach to find potential quantitative differences in peak intensity between case and control sera. To simplify data, 2-min elution windows were defined. It was critical to do quantitative analysis to correct for day-to-day and run-to-run cLC variation. While elution order and relative elution times remained fixed, changes in elution start times for individual serum runs varied. To insure coincident time alignment of spectra, 10 endogenous serum peptides that eluted at ~2-min intervals throughout the chromatogram were selected as reference peaks. Referencing to these control peaks’ elution times allowed for successful alignment of each 2-min window and for species across the entire elution interval, making possible more accurate comparisons of potential quantitative differences between cases and controls. The first peptide used for time alignment has been identified as fibrinopeptide A. This simple method of time normalization allowed for appropriate overlay of case and control spectra, making possible manual serum biomarker discovery.
Recently, more emphasis has been placed on developing validated SRM methods for performing targeted protein quantitation. The key to successfully quantifying the protein expression level is to select peptides that are unique to the targeted protein, yet provide enough sensitivity to detect in a complex background matrix like plasma. Newly presented strategies rely on searching the precursor/product ion m/z values with similarly created database pairs to determine whether the SRM transition is unique in the context of the background matrix. This, however, may eliminate a potentially powerful proteotypic peptide from a validated assay, if the selected mass pair is common. This strategy may also eliminate the most sensitive transition, because it did not have enough selectivity. Our approach is motivated by the belief that for successful quantifying of extremely low-abundant proteins, we need to achieve the highest levels of sensitivity possible without sacrificing selectivity. In this work, we provide a computational strategy to determine for a set of proteins, which peptides and corresponding transitions provide the best sensitivity and selectivity to confidently identify the correct retention time and response for protein quantitation. For some peptides, we might need only one transition, whereas for some peptides, two or more transitions may be required. To test out the new algorithm, 100 enzymatic peptides are selected from bovine plasma proteins and processed. The peptides are selected based on identification from previously acquired discovery experiments and are separated into categories based on peptide length. The ranking of the SRM transitions for each peptide are determined based on linear ion trap CID and orbitrap HCD experiments. The most abundant transition is processed to determine its selectivity factor and need for additional transitions to ensure its uniqueness within the background matrix.
The application of MALDI mass spectrometers to determine the spatial distribution of endogenous and exogenous chemical species in tissue is a rapidly developing area of research. A promising branch of this technique is the study of the distribution of metabolites. MALDI imaging has great potential, offering complementary information to traditional techniques.
The two main instrumental challenges for the mass spectrometric analysis of these types of samples are sensitivity and specificity, i.e., how well the compound of interest can be distinguished from background ions.
The efficient detection of small molecules in a biological matrix is often difficult owing to competing background ions from the tissue. This effect is exacerbated in the case of MALDI imaging by the presence of the matrix. We introduce high-definition mass spectrometry (HDMS) combined with MALDI imaging. HDMS offers a new dimension of separation, combining high-efficiency ion mobility separation (IMS) with orthogonal TOF mass spectrometry. Separation of ions according to size, shape, and charge state is possible. Using this technique, it is possible to separate different compound classes.
The sample studied was a thin section of rat kidney. A 12-μm section was produced using a cryotome, and deposited on thick aluminium foil. An airbrush was used to apply several coats of α-cyano-4-hydroxycinnamic acid matrix evenly to the sample. The sample was mounted on a target plate. The area to be imaged was selected and data were acquired on a MALDI Synapt HDMS system operated in mobility TOF mode. After acquisition, HDMS data evaluation was performed. Data were converted into Analyze file format, and image analysis was performed using BioMap.
We show data demonstrating the separation of nominally isobaric species using IMS in an imaging experiment, producing distinct images for the separated compounds.
Clinical routine diagnosis of biomarkers is mostly based on immunological, quantitative techniques—e.g., ELISA. These methods are often not applicable for small antigens or for antigen isoforms. MALDI-TOF MS is a simple, sensitive, and rapid method for detecting peptides and distinguishing different protein/peptide isoforms. Hence, quantitative mass spectrometry is a desirable alternative diagnostic tool. The infeasibility of absolute quantification is a tremendous handicap hampering stable clinical diagnostics. We have developed a technical platform based on ClinProt immuno-particles and heavy internal peptide standards, combining the advantages of MALDI-TOF MS as a read-out system and absolute quantitation of peptide biomarkers.
For absolute quantitation of peptides, the natural peptide and a corresponding counterpart comprising heavy amino acids were synthesized, resulting in two peptides with similar chemical/biochemical properties and a defined mass difference. A peptide-specific antibody was covalently coupled onto magnetic particles, and these were employed for the isolation of both peptides. A calibration curve was acquired by mixing the peptides at different ratios, keeping the concentration of the heavy peptide constant. After immuno-capturing, the ratio of the peak areas of both peptides from the MALDI-TOF spectrum was plotted against the concentration of the natural peptide. To determine the concentration of the peptide of unknown samples, the heavy peptide was spiked. Purification of samples was performed by ClinProt affinity particles, and peak areas of the subsequent MS analysis were calculated.
The presented work was set up as a proof-of-concept study for absolute quantification by MALDI-TOF in a clinical setting. As a model system, des-Ala-fibrinopeptide A (des-Ala-FPA) and a corresponding antibody were employed to investigate the absolute concentration of des-Ala-FPA. A kinetic analysis of the influence of temperature by time on the des-Ala-FPA concentration demonstrated a rapid degradation of the peptide within minutes.
A junction-style high-voltage contact at the column inlet is a robust and facile configuration for nano-LC ESI-MS analysis. When seamlessly integrated onto the nanobore column, chromatographic performance is maintained with minimal contributions to band broadening. When the voltage is applied post-column, chromatographic performance is compromised, resulting in band broadening and post-column loss, due to the internal volume of conventional unions. To achieve optimal chromatographic performance, scientists strive to minimize and ideally eliminate all sources of dead volume within their LC systems. An electrically conductive, optically clear, zero-dead-volume union offers promising solutions for a post-column high-voltage junction contact. The superior performance of an electrically conductive zero-dead-volume union, compared to conventional post-column high-voltage contact unions, is demonstrated by chromatographic performance improvements in analyzing a BSA tryptic digest. Further applications of this novel conductive union are discussed.
Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules such as proteins, peptides, oligonucleotides, and oligosaccharides, which tend to be fragile and fragment when ionized by more conventional ionization methods. MALDI analysis is particularly suitable for high-throughput identification of proteins through analysis of low-complexity peptide mixtures, such as in peptide mass fingerprinting or in MS/MS analysis of digests of single proteins or simple protein mixtures. These applications benefit substantially from high-accuracy mass measurement, as high mass accuracy increases the number of confident identifications and percent sequence coverage, particularly as sample complexity increases. Here, we examined the mass accuracy, sensitivity, and peptide fragmentation performance of an LTQ Orbitrap XL instrument operating with a MALDI ion source. The instrument was found to consistently display high mass accuracy, with error on the order of 0.5–2 ppm. Sensitivity was found to be very high using short analysis times. For example, 5 fmol of digested BSA were analyzed using five FTMS scans. PMF analysis of the resulting data gave 50% coverage of the protein, allowing confident identification. We examined peptide fragmentation performance for singly charged ions using this instrument’s Higher Energy Collisional Dissociation (HCD) mode. This function was found to give productive fragmentation of single charged ions, and displayed superior performance with phosphopeptides as compared to CID.
Sustainability of emitter viability and analytical performance remains a salient objective for continuous nanobore ESI-MS experiments. Several days’ extended use can engender debris accumulation at the emitter tip, Taylor-cone impedance by flow-path obstruction, and subsequent analyte-ionization deterioration. Fused-silica electrospray emitter tips coated with low-density polyethylene (LDPE) have displayed exceptional resistance to debris accumulation after several weeks’ continuous infusion with acetonitrile-extracted canine plasma. When detached and repurposed for continuous-infusion electrospray analysis of a 1 pmol/μL Angiotensin II solution, the same LDPE-coated emitters display enhanced spray stability and ionization fidelity in weekly nESI-MS analyses.
Irinotecan has shown strong antitumor activity against a wide variety of human solid tumor xenografts in mice. Irinotecan is a DNA topoisomerase I inhibitor that binds to and prevents dissociation of the DNA-topoisomerase I complex (involved in DNA replication), thereby inhibiting enzyme activity.
Lack of microvessels has been shown to offer resistance to irinotecan therapy in human head and squamous carcinomas. Two types of model tumors, FaDu and A253, with high vascular and avascular regions, respectively, were used in a previously published study. The purpose of our study is to demonstrate the utility of imaging mass spectrometry for looking at drug distribution in FaDu tumors as compared to healthy liver and establish MALDI MS–based imaging as a viable technique in cancer therapy research.
Nude mice with implanted human head and neck tumors were treated with either irinotecan, methylselenocysteine (MSC), both drugs combined, or non-treated (control). Sectioned tissue (12 micron) was thaw-mounted onto non-conductive glass slides and MALDI matrix sprayed on top. Irinotecan metabolites were investigated with an LTQ XL coupled to a MALDI source. The acquired data were analyzed with in-house software to visualize the distribution of drug within the tissue and tumors.
Irinotecan was detected in the dosed liver and tumors by imaging mass spectrometry. Both the parent drug (m/z 587) and known metabolites SN-38 (m/z 393), SN-38-glucuronide (m/z 569), and M2 (m/z 603) were detected by MS2 and MS3 tandem mass spectrometry in positive ion mode. MS3 spectra were more specific, showing only metabolite fragments with no contribution from lipids or matrix adducts. Preliminary images show more uniform metabolite localization for the tumors treated with both irinotecan and MSC, but apparent higher concentration of metabolite in the tumor treated with irinotecan by itself. Tissue replicates will be analyzed next to determine consistency of preliminary results.
Electron transfer dissociation (ETD) is the preferred method for the analysis of post-translational modifications (PTM) in proteins. It surpasses internal heating and generates rapid N-Cα bond cleavage along the amino acid backbone, leaving labile bonds like those to PTMs intact.
Proton transfer reaction (PTR) is a most useful addition for the fragmentation of larger peptides or small proteins in the top-down or mid-down approach. Following the ETD reaction, PTR anions reduce the charge states of the highly charged fragments into ion trap readable numbers.
For ETD, an excess of radical anions generated in a negative chemical ionization (nCI) source is added to multiply charged peptide cations in the ion trap. Fluoranthene is a commonly used ETD reactant. While this reactant is a strong electron donor, the PTR reactant must have a high proton affinity for efficient proton stripping. Therefore, different reactants have been proposed, such as benzoate or PDCH. Handling several reactant reservoirs, plus different nCI sources, becomes complicated and prevents effective MS/MS automation.
Proposed here is a system with a single nCI and fluoranthene as the only reactant. For ETD, fluoranthene itself is applied. For PTR, a naphthalene-like fluroanthene side product generated by modifying nCI parameters is used. A fast and automated switch-over between ETD and PTR is possible within a few msec.
The 3D trap itself has a number of additional inherent features for fast and effective ETD/PTR: The simultaneous storage of cations and anions provides for fast and direct ETD.
Cations and anions are mixed and compressed in a small globular volume, resulting in an excellent ion-ion cross-section and highly efficient ETD.
The high m/z range of the 3D trap of 3000 U with sufficient mass resolution is essential for PTR of proteins.
Those features will be demonstrated with typical proteomic samples for PTM elucidation and top-down identification.
Quantitation is increasingly important in proteomics. In addition to compound identification, researchers are increasingly interested in relative protein abundances between reference and case-study samples that may give insight into the protein functionalities in relation to distortions of the system like diseases, stress, functions, etc.
Proteins found in the discovery phase are called bio-marker or drug-target candidates. Candidates need to be validated in a second stage of the discovery process. In contrast to the discovery, which is a global approach, the validation of candidates allows a rather focused—or targeted—approach. Only the candidate proteins are considered in this validation process, and any additional information produced at the same time is ignored. Most important for this validation step are quantitation accuracy, analysis speed (as biological and technical replicates need to be considered), and analysis cost (as many samples are typically quantified for candidate validation).
The multiple reaction monitoring (MRM) method in triple quads is a most cost-effective and fast approach for targeted quantiation. In MRM, Q1 is tuned in on a known peptide parent mass, Q2 on the efficient fragmentation, and Q3 on a specific fragment mass. The mass chromatogram of that specific parent-to-fragment transition is used for quantitation.
Instead, here a label-free approach with an ESI-TOF was used. Due to the mass accuracy of the ESI-TOF (independent of the peak intensity), it is possible to generate high-resolution extracted ion chromatograms (hrEIC) with a mass tolerance window of ca 2 mDa. This allows for an outstanding selectivity of compounds vs. background. Those highly selective hrEICs can be used for quantitation. Advantages of this novel concept are the unlimited number of target ions as well as the possible revision of data to quantify additional peptides, if needed at a later stage after the run.
The study of human health and metabolism is the focus of research undertaken at the Human Metabolic Research Unit (HMRU). The HMRU is located in the Division of Nutritional Sciences at Cornell University and provides a state-of-the-art research infrastructure for investigators who conduct research with human subjects. This unit contains multiple resources to accommodate topics relevant to human metabolism, nutritional biochemistry, clinical chemistry, body composition, and dietetics. Access to the HMRU facilities is open to both researchers and those individuals who are interested in assessing their own physical performance and health status.
Chronic rejection (CR) remains a major cause of allograft loss following kidney transplantation. Current biomarkers for kidney function are relatively insensitive and nonspecific. The objective of this study is to develop a method to comparatively profile metabolites in urine of rats that develop CR with rats where effects of CR are attenuated by immunosuppressants. We postulate that differences in metabolic profiles will correlate with progression towards CR and that, ultimately, a panel of metabolic biomarkers can be validated to assess CR in human transplant patients.
Lewis rats served as recipients in a life-supporting kidney transplantation of Fisher 344 kidneys. Group 1 (n = 10) was treated with cyclosporine (1.5 mg/kg BW/d) for 10 d to prevent or reverse acute rejection, followed by daily treatment with vehicle. Group 2 (n = 10) was treated with cyclosporine (3.0 mg/kg BW/d) for 30 wks. Recipient rats were restrained in metabolic cages, allowing collection of 24 h urine at various time points. Proteins were precipitated and metabolites separated by HPLC with a gradient of 0.1% TDFHA and acetonitrile. Eluting compounds were analyzed using ESI-TOF-MS (Agilent). Molecular features were extracted and compared with MassHunter software. Signals from metabolites showing greater than 2σ difference in intensity were selected for further investigation.
Twelve features were found to be significantly different between the two groups. Database searching of accurate masses elucidated potential metabolites. One of the features found increased in Group 1 was methyl 3-isothio-cyanatopropionate. Amcinonide was increased in Group 2.
Our results suggest that different cellular defense mechanisms are important in the two treatments. The isothiocy-anatopropionates (increased in Group 1) have anti-oxidant activities, and amcinonide (increased in Group 2) is a corticosteroid with anti-inflammatory properties. Results of this preliminary study suggest that metabolic profiling can add to our mechanistic understanding of allo-recognition and chronic rejection.
Transgenic modification of crop plants to improve product quality or agronomic performance is a commonly utilized tool for modern agriculture. However, consumer concerns regarding transgenic crops has restricted the use of plant transformation from many sectors of agricultural production. Part of the consumer aversion is due to fears of unintended effects on plant composition or development due to plant transformation or other stochastic factors. Two important concepts in the debate over transgenic crop safety are substantial equivalence (SE) and generally regarded as safe (GRAS). SE is the concept that a transgenic variety is so highly similar to its nontransgenic parent that it can be considered to be the same. Opponents of transgenic crop improvement have criticized this concept for being without statistical merit or utility for risk assessment. In the plant improvement context, GRAS means that we accept that the products of conventional plant breeding (i.e., new varieties) are safe. Thus, the differences that exist between conventionally improved plant cultivars represent a threshold that is acceptable to consumers, regulators, and other stakeholders. We will assess the differences between conventionally and transgenically modified tomatoes with altered fruit ripening using nondirected metabolomics. Our emphasis will be on the ripening inhibitor (RIN) mutant and transgenic approximations thereof. Wild-type RIN is a MADS-box transcription factor necessary for ethylene-dependent ripening in fruit; mutant rin plants lack this signaling pathway and thus a major ripening promoter. The rin mutant is in widespread use for tomato improvement by public- and private-sector breeders.
Providing unprecedented accuracy and robustness in elemental speciation, speciated isotope dilution mass spectrometry (SIDMS) (U.S. EPA Method 6800) is supported by 10 years of scientific scrutiny and scholarly publications since its adoption in 1998.
The unique dual-species or three-species, ratio-based, simultaneous chemical and computational measurement approach utilized by Method 6800 eliminates the need for calibration, saves time and money, and reduces inaccuracies from instrument signal drift. The results are deterministic and legally defensible.
One of the obstacles that limited greater acceptance of this powerful method have been unavailability of standards and reagents, and a convenient, straightforward way to prepare and analyze samples in matrices that range from soil, sediment, water, petroleum, industrial products, and biological specimens. Although some of the challenges were addressed with the availability of new standards and speciation kits, the expanded Method 6800 is accepted for promulgation, and the new globally adopted environmental laws such as Restriction of Hazardous Materials (RoHS) (adopted by the European Union) include required testing for metal species, there are still formidable challenges before speciation becomes a routine method.
Achieving true quantitation of dynamic species is essential for a reliable, accurate assessment of toxicity. The importance of accurate metal and mineral species measurement is underscored by an increasing number of biomedical and environmental health papers that identify environmental exposure as one of the critical factors acting as triggers and accelerators for diseases like cancer and autism. Early studies hold great promise, but there is still much work ahead. This poster will include data from recent unique environmental health applications and address key areas that need improvement before SIDMS can turn into a mainstream tool across disciplines and industries.
The most generic method applicable to both proteomics and metabolomics is nontagging quantitative profiling. The primary benefit of this form of assay is provided by its generic nature—it can be used for both metabolomic and proteomic applications. In this poster, we present the use of such a generic method for the analysis of both types of samples and use a generic software application of the feature finding and differential analysis.
In the proteomic experiment, two complex mouse muscle lysates were processed using a nanoflow LC system and 100-μm Merck monolithic column. Peptide detection was performed using an orthogonal time-of-flight mass spectrometer. Initially, each sample was processed five times using repeat information-dependent acquisition to provide an accurate picture of the sample. In the metabolomic experiment, the same LC and mass spectrometer setup was used. A multipoint urine study was undertaken, with each sample injected multiple times. The data from both experiments were then processed with MarkerView software to identify species whose levels are modified.
Either complete feature extraction or targeted feature extraction was used to analyze the differences between the samples by t-test and principal component analysis. Identical statistical tools and software were used to process the divergent sample types. In the case of the metabolomic experiment, a target include list was produced ready for compound identification, while in the proteomic experiment, either the identification was performed prior to statistical analysis or the MS/MS of the different feature was searched to identify the species. This highlights that the divergent applications of metabolomics and proteomics can be treated in identical manners and data processed in a similar fashion.
Untargeted metabolomics workflows typically include sample preparation, data collection, feature finding, statistical analysis, and compound identification. In an effort to simplify and streamline the compound identification step in a high-throughput manner, we have collaborated with the Scripps Research Institute to create a customizable personal metabolite database using content from their Web-based METLIN Metabolite Database. This is the largest database in the world, and includes masses, chemical formulas, and structure information for over 15,000 metabolites. Retention time (RT) and an automated molecular formula generation (MFG) capability have been added, which use isotopic pattern matching to enable higher confidence identification of compounds from accurate mass data. It can be used concurrently with database searches, generating a list of masses with a specific RT, a metabolite compound match, and a putative formula(s). Thus MFG provides additional confirmation of the compound identification generated by the database search. Our personalized database also enables batch search querying of an unlimited number of stored masses and associated RTs. For masses that do not have a database match, the molecular formula generated can still be used to guide further experiments for compound identification. The software has the capability of creating custom databases to which new compounds can be added. A urine database was developed from a mixture of standards representing the most abundant compounds normally found in urine, to generate a list of masses and associated RTs using LC/ESI time-of-flight mass spectrometry in positive ion mode. After the database was populated, actual urine samples were analyzed under the same analytical conditions and screened for mass and RT matches against the urine database of standards. We used MFG to confirm the results of the database matches. Identified metabolites were annotated with a chemical formula, structure, and other metadata, including CAS and KEGG identifiers
Secondary metabolites derived from bacterial extracts are important sources of potential new drugs. High-resolution chromatography and mass spectrometry are powerful tools for the analysis of highly complex mixtures. As manual data examination is often not feasible due to complexity and sample number, statistical methods can facilitate the extraction of relevant information. A metabolomics-based approach for myxobacterial extracts is presented, using ESI-TOF-MS data, principle component analysis (PCA), and a subsequent identification.
Bacteria were fermented in a 50-mL scale using complex media and XAD-16 resin as absorber. LC-MS measurements were performed using an orthogonal ESI-TOF mass spectrometer coupled to a fast HPLC system equipped with photodiode array detector. Data were acquired in ESI positive and negative mode from 150–1200 m/z. Sodium formate solution was injected as external calibration standard. Data were evaluated using ProfileAnalysis software for PCA and molecular formula generation.
Differences in production patterns of myxobacterial strains can be extracted by PCA from LC-ESI-TOF-MS data, even considering significant biological variation within replicate fermentations.
The variation within the fermentation experiments was assessed for five closely related strains with six biological replicates.
LC-MS data were prepared for PCA analysis in Profile-Analysis software. The replicate extracts revealed considerable deviation within replicate fermentations, but still resulted in a clear formation of groups. Compounds representing strain differences were identified by both accurate mass and isotopic pattern. The variability of the instrumental analysis was regularly checked by two different quality control samples.
In a second experiment, 120 bacterial isolates of the species Myxococcus xanthus from places around the world were cultured and analyzed. PCA was applied to find trends in different production patterns, which may be correlated with the geographical distribution of the samples, but also biological properties of the strains. Accurate mass data were applied to identify putative novel metabolites.
Microchip Biotechnologies is developing a paradigm-shifting microfluidics-based product, the Apollo 100, for integrating, automating, and simplifying the process of Sanger sequencing. The Apollo 100 combines a proprietary microchip thermocycling system, the Apollo 100 Chip Station and microfluidic chips, a small-footprint liquid handling robot, and user-friendly software to control all components without the need for custom scripting. At the heart of this product is our patent-pending Microscale On-Chip Valves (MOV) with on-board pneumatics and thermocycling control systems, providing an efficient mode of mixing, moving, and cycling sequencing reactants on chip. The Apollo 100 process is simple. DNA template and reagents are loaded robotically onto four 24-channel microchips; the pneumatically activated MOV valves distribute, mix, and automatically set up dye-terminator sequencing reactions. After cycle sequencing, on-chip bead purification produces ready-to-inject processed samples that are robotically transferred to a multi-well plate for analysis on commercial capillary array DNA sequencers. The Apollo 100 chip, chip cycler, and robot are controlled by the Apollo 100 Operating Software, which is driven by Microchip Bio-technologies’ proprietary DevLink software. The Apollo 100 reduces the cost of DNA sequencing while maintaining the quality of results by integrating and automating the processes of sequencing reaction setup, cycling, and cleanup in a microfluidics environment. The Apollo 100 reduces the amounts of sequencing reagents necessary for running efficient reactions and removes most of the manual steps, allowing the user to place samples and reagents on the instrument, walk away, and return to sequencing products that are ready to inject onto a capillary sequencer. Performance data for read-length and signal-to-noise for an Apollo 100 prototype system that processes 96 samples simultaneously are equivalent to full-volume reactions.
Cytochrome P450 and monoamine oxidases are two groups of enzymes involved in the metabolism of drugs. The interactions of these enzymes and compounds are studied carefully in early the drug discovery process to facilitate greater success in clinical trials. Promega luminescent metabolism assays consist of the P450-Glo Screening Systems for CYP 1A2, 2C9, 3A4, 2C19, and 2D6, as well as the MAO-Glo Assay System for monoamine oxidase A. Using the Labcyte Echo 550 acoustic liquid handler and the Deerac Fluidics Equator HTS reagent dispenser, a collection of compounds was profiled by IC50 against the panel of metabolism assays in 1536-well format. Total assay volume of 5 μL was achieved by combining the acoustic technology and noncontact spot-on technology. The flexible incubation times of all assays, in addition to the single luminescent endpoint readout, allowed the assays to be run on the same plate and simplified data analysis. Profiling with IC50 determined both potency and selectivity of the test compounds against selected enzymes involved in the drug metabolism process. The exceptional Zk Factor scores, show the flexibility and reliability of Promega assays in conjunction with Labcyte Echo and the Equator HTS from Deerac Fluidics in a high-throughput situation.
G protein–coupled receptors (GPCRs) are involved in various physiological processes, such as the regulations of behavior, mood, and immune system activity. GPCRs are popular targets in drug discovery, and a well-designed assay can speed up the discovery of novel drug candidates. The Promega cAMP-Glo Assay monitors cyclic AMP (cAMP) production in cells in response to the effect of an agonist or test compound on GPCRs. The assay is based on the principle that cAMP stimulates protein kinase A holoenzyme activity, decreasing available ATP and leading to decreased light production in a luciferase reaction. Together with the Labcyte Echo 555 acoustic liquid handler and the Deerac Fluidics Equator HTS reagent dispenser, compounds can be screened in 1536-well format for effects on GPCRs. Implementing a high-throughput miniaturized GPCR assay as demonstrated in this poster allows cost-effective screening for the next blockbuster drug.
Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting towards the use of cell types of increasing scientific interest, including lineage-specific cell types, bone marrow cells, and stem cells, but these cells can be difficult to transfect. The ability to transfect these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability would offer a new tool towards the elucidation of the pathway(s) in which a gene product is involved.
In this poster, we present data demonstrating that optimization of electroporation parameters is essential to deliver molecules in a highly efficient manner. We display results of transfection of several difficult to transfect cell types including human primary fibroblasts (HPF), human umbilical vein endothelial cells (HUVEC), Jurkat cells, and (mouse) Neuro-2A neural cells with plasmid DNAs and siRNAs. We used multiple detection methods to determine transfection efficiency, including flow cytometry, real-time PCR, and the luciferase assay. Gene Pulser electroporation buffer and square or exponential wave pulses were used to convey molecules of interest into the cells. Optimization of electroporation parameters was carried out using the high-throughput Gene Pulser MXcell electroporation system. Just 4 h post electroporation with a GAPDH siRNA, Jurkat cells expressed 88% less GAPDH mRNA than controls transfected with scrambled siRNA. The same degree of silencing persisted at 24 and 48 h post electroporation. Transfection of cells with fluorescently labeled siRNA resulted in 75% transfection efficiency for Neuro-2A, 93% for HPF, and 98% for HUVEC cells, as analyzed by flow cytometry. We provide optimized electroporation conditions for these and other difficult to transfect cells.
MicroRNAs (miRNAs) are short, 20–22-nucleotide RNA molecules that have been identified recently as sequence-specific regulators of many cellular processes such as apoptosis, proliferation, and differentiation. Meanwhile, hundreds of miRNAs have been discovered in the genomes of animals and plants, but they are only beginning to be classified by their functional roles. One of the major drawbacks is the lack of adequate analytical methods for the analysis of small RNA samples. Here, we compare different methods for analysis of small RNAs from blood samples.
The use of RNA interference (RNAi) in research offers a powerful tool to modulate gene expression and determine gene function in mammalian cells. The activation of an RNAi pathway can result in the sequence-specific degradation of a messenger RNA (mRNA) and reduction of its corresponding protein product. Key considerations for a successful RNAi experiment are: (1) the use of an effective siRNA silencing molecule, and (2) delivery of silencing molecules with high transfection efficiency and low cytotoxicity.
Recent findings demonstrate that a new class of double-stranded RNA molecules with increased length and altered end structure are up to 100-fold more potent at gene silencing when compared to standard siRNAs. When coupled with a robust delivery method, this potent silencing has been shown to be compatible with a variety of cell lines, using conditions and strategies common to RNAi experiments.
Recent studies have demonstrated that 27-nucloetide-long duplexes (known as dicer substrate siRNAs) enter the natural RNAi processing pathway at an earlier step and may be more potent activators of the RNAi pathway in cultured mammalian cells. Remarkably, it has been demonstrated that these longer 27mer siRNA duplexes do not activate the protein kinase R pathway, nor do they induce an interferon response. These siLentMer siRNAs are capable of silencing genes for a longer length of time when compared to standard siRNAs—the result of an enhanced interaction with RNA endonuclease Dicer. The siLentMer siRNA duplexes deliver highly potent gene silencing, even at concentrations as low as 100 pM. These siRNA duplexes also reduce potential off-target effects, and have been functionally tested via RT-qPCR to guarantee >85% reduction in target mRNA levels. These results were generated using siRNA for a variety of targets. Bio-Rad’s siLentFect lipid reagent was also used to demonstrate highly efficient transfection of siRNA with extremely low cytotoxicity.
Modulating gene expression is essential to achieve a better understanding of gene function. The transfer of exogenous nucleic acids such as plasmids or small interfering (si) RNAs into mammalian cells is an important technique for the study and analysis of gene function, expression, regulation, and mutation, and it has advanced basic cellular research, enabled drug target identification, and validation. Electroporation is a well-established gene transfer method, and an effective means of transferring nucleic acids into cells. The shift of today’s research to more scientifically relevant cell lines has created a need for efficient delivery tools. In addition, the wide adoption of RNAi technology, has led to a demand for screening tools to identify the most effective siRNA. In order to obtain consistent downstream results, it is critical that delivery conditions be highly efficient.
Here, we show data that demonstrate the benefits of using a high-throughput electroporation system to develop optimal conditions for delivery of plasmids and (si)RNA into mammalian cells . We demonstrate that the use of this instrument allows for rapid screening as well as the ability to identify the most efficient delivery conditions into a difficult-to-transfect cell line. Finally, we present quantitative PCR data that validate the benefits of this technique.
The Wadsworth Center is the most comprehensive state health laboratory in the country, and is dedicated to science in the pursuit of health. It fulfills its mission of protecting and promoting the health of New Yorkers through analysis, research, and education combined with comprehensive clinical and environmental laboratory evaluation and accreditation programs. Our scientists study public health issues, from drug resistance to emerging infections and environmental toxicants, as well as basic biological processes.
The Core is a resource for investigators within the Wadsworth Center as well as those from outside institutions who require advanced light microscopic (LM) and/or image analysis as part of their research programs. Our goal is to operate a user-friendly, full-service Core for our user community, including a comprehensive educational program. This includes the latest in imaging equipment and methods, and in image analysis software and methods. In the event that current instrumentation cannot answer the question posed by the user, we seek to develop innovative solutions. One example of this is optical projection tomography; this modality bridges the resolution gap between NMR/CAT scans and traditional LM.
Whatever the specimen or the question(s) being asked we provide the technologies and assistance to get the answers. A few recent examples are: Imaging waterborne contaminants to identify the organism responsible for sickening the patrons of a water park. Imaging motile bacteria at near video rates and then tracking them to investigate the cellular effects of an antibiotic. Visualizing multiple biomolocules/fluorophores in 3D, to investigate neuronal function in brain slices. Automatically counting cells outgrowing from explants in order to map quantitative trait loci between inbred strains of mice in response to vascular endothelial growth factor. Measuring the fluidity of lipid bilayer using FRAP to determine how lipid mobility controls the diffusion of small biopolymer adsorbates.
Sample preparation is crucial for the quality of MALDI tissue-imaging data. Unfortunately, the current matrix application protocols have significant disadvantages: While pneumatic spray preparations provide good homogeneity and spatial resolution of the images, the process is manual and not highly reproducible. Depending on the degree of tissue wetting, either the analyte molecules are badly incorporated into the matrix (too dry) or the spatial resolution is lost (too wet). Nano-spotting, on the other hand, provides quality spectra, but, as a sequential process, it is slow, spatial resolution is limited by the spot raster (typically >200 μm), and perfect alignment with the mass spectrometer is critical. We introduce an entirely new approach that combines the advantages of these methods and eliminates the disadvantages. In the new preparation device, a matrix aerosol (20-μm droplets) is created by vibrational vaporization under controlled ambient conditions and is gently deposited onto tissue sections. Tissue sections can be homogeneously matrix coated, typically with 30–100 deposition-incubation-drying cycles within one hour. An optical sensor monitors scattered light from the matrix layer, allowing us to control all relevant preparation parameters in real time: deposition periods, intervals, matrix layer thickness, wetness, drying rate. This sensor control of the sample reproducibly provides a wetting/crystallization regime that is prerequisite for achieving a high image resolution (up to 25 μm) and high spectra quality at the same time. This high MALDI image accuracy gives high resolution and high accuracy matching between histological/ immunohistological images, essential for many molecular histology applications.
MALDI imaging is a novel technique providing unique molecular information about histological tissue sections. We applied MALDI imaging at high matching accuracy between MALDI images and histological images to a set of tissue sections from breast cancer patients to develop automatic tissue classification routines.
The analysis of larger sample cohorts, such as breast cancer patients, requires statistical analysis of resulting MALDI imaging data. Here, we applied different statistical tools, such as unsupervised principal component analysis (PCA) and hierarchical clustering, and supervised classification algorithms (SVM) to access histological information from the MALDI imaging data in a largely automatic fashion.
Unsupervised PCA analysis allowed the direct visualization of the variance in the MALDI imaging datasets. In most cases the PCA results were in good correlation with the histological examination of the sections. In some cases, however, the results of the PCA did not correlate with the histology. This was due to intensive signals from compounds such as beta-defensins, which originated from contamination from blood. Exclusion of such peaks from the PCA gave the expected results. Unfortunately, the PCA resulted in a high variance if tumor sections came from different patients. Therefore, the PCA reflected largely the variation across patients rather than variation across tissue types. In contrast, SVM gave direct access to molecular species that were characteristic for specific tissue types. The visualization of the classification results as a 2D image (class imaging) also facilitated the comparison with immunohistostaining. Using the supervised classification approach, it was possible to create a software model for the classification of HER2-positive cancer, HER2-negative cancer, and connective tissue. It was possible to apply this model to unknown tissue sections to obtain the correct, simultaneous classification of both tumor types, as confirmed by the inspection of the pathologist! However, larger studies are required to provide a final validation of this approach.
Ion exchange (IEX) chromatography has emerged as a reliable alternative to classic CsCl gradients for isolating nucleic acids of the highest purity. Edge PurElute IEX plasmid purification was developed for the production of superior quality plasmids. Plasmids are extracted from bacterial cells by a modified alkaline lysis method. The crude lysate is cleared by centrifugation (MiniPrep) or by syringe-format filtration (MaxiPrep). Prior to IEX chromatography, an extraction solution is added into the clarified lysate. The mixture is loaded into a PurElute spin column without the need for extra incubation and separation. Plasmids are captured, while bacterial contaminates such as endotoxins are prevented from binding by the extraction solvent. Binding, washing, and elution are optimized to achieve efficient isolation of plasmids from the impurities. After precipitation with isopropanol and washing with ethanol, plasmids are dissolved at concentration as needed for downstream applications.
PurElute IEX spin columns are made of a unique anion-exchange matrix, which has an exceedingly high binding capacity. The capacity for binding plasmid is at least 50 μg for a MiniPrep column and 1 mg for a MaxiPrep column. The columns are ready to use without the need for an equilibration step. Residual solution in column can be spun down nearly completely, resulting in a clean separation of impurities, reduced buffer volume for each step, and high recovery efficiency. DNA can be eluted in a relatively small volume, i.e., high concentration, which makes isopropanol precipitation much more efficient. In most cases, DNA pellets are visible, lowering the risk of losing sample. Purified plasmid is free of impurities that may interfere with critical downstream applications, e.g., mammalian cell transfections. Performance of PurElute IEX plasmid purification was evaluated with gel analyses and endotoxin tests, with comparison to other commercial products.
Recent advancements in assay miniaturization have combined advanced molecular biology analysis methods with droplet-based microfluidics technology to enable a vast number of analytical reactions (10,000 samples/sec) to take place using extremely low volumes of samples and reagents in microscopic droplets.
We present the application of this new method of fluid-handling technology to areas including flow cytometry and multiplex PCR. In the first example, we present the use of droplet-based flow cytometry for detection of low copy number cell-surface biomarkers, demonstrating greater than 100X enhancement in signal:noise relative to traditional flow cytometry techniques.
In the second case presented, droplet-based primer libraries are used to amplify regions of for targeted genome sequencing by highly multiplex PCR reactions. The novel approach to multiplex PCR enables amplification of thousands of unique amplicons in a simple and straightforward experimental design.
Sequencing reaction purification (SRP) is normally carried out using gel filtration, solid magnetic substrate, membrane platform, or standard ethanol precipitation to eliminate salts, unincorporated dNTPs, and dye terminators from the extension products. However, these methods all require centrifugation, which hinders the ability to effectively automate SRP. We have developed a surface-modified microplate to perform SRP without the need for centrifugation. All manipulations can be performed on a robotic platform, making this technology very amenable to automation. The extension products are selectively adsorbed to the surface-modified microplate, washed, and eluted in water. Furthermore, this new technology not only offers a robust, high-capacity, and fully automatable SRP platform, but also works efficiently with reduced volumes of BigDye terminators.
Testing was done on a Biomek FX workstation with a 96-well head. Extremely reproducible results over a wide range of reaction conditions were achieved in about 30 min. The system was evaluated in 384-well plate configuration on an Applied Biosystems 3730xl DNA Analyzer using the ABI PRISM BigDye Terminator v3.1 chemistry. A variety of conditions were interrogated, with sequencing reaction volumes ranging from 2 to 6 μL, DNA template masses ranging from 50 to 100 ng, and BigDye volumes ranging from 100 to 330 nL. On average, typical Phred20 scores of 800 were achieved.
The extraction and purification of nucleic acids prior to performing genomic analysis is a critical step in the generation of gene expression data when using microarray platforms. Several studies have compared expression profiles using total RNA or mRNA prepared from the same sample. The historical data largely reported on comparable results between these two approaches and concluded that comparative data analysis resulted in similar, but not necessarily identical, gene expression profiles.
This study revisits this issue, taking a more detailed view of the observable differences between the two approaches. The study comments on under which circumstances the use of mRNA may be a preferable choice for target preparation compared to the more usual preparations of total RNA.
The Vermont Cancer Center DNA Analysis Facility provides an array of fast, affordable, user-friendly DNA analysis services to the members of the Vermont Cancer Center and the University of Vermont research community. Primary services offered for a fee include DNA sequencing, real-time quantitative PCR, DNA fragment analysis (microsatellite instability, haplotyping, AFLP, T-RFLP, and SNaPshot), SNP detection, nucleic acid extraction, and image analysis (phosphorimaging, fluorescent detection, DNA and protein stain imaging, chemiluminescence, and densitometry). Our newest service is protein gel spot excision with a BioRad EXQuest Spot Cutter. The facility provides a variety of other important benefits free of charge to our users. These include sponsoring seminars on new technologies, holding tutorials on software and user-driven instrumentation, primer and probe design, obtaining discounted pricing on common reagents, and providing data analysis, troubleshooting, and consultation in experimental design. Our annual open house is a popular day-long event highlighting all of our services. In January 2007, the facility began a new User Educational Seminar Series with the goal to meet with users on a monthly basis to educate and promote services currently being provided in the facility. A computer workstation area is adjacent to the main lab and has freeware and purchased programs available for data analysis and review with full support from nearby facility staff. The facility uses a bioinformatic platform known as the UVM BioDesktop to facilitate the workflow for both the users and the staff of the facility. Users of the facility create a password-protected BioDesktop account for online ordering of all services, viewing and archiving data, and accessing data-analysis tools. The facility staff uses their BioDesktop account for uploading completed data, creating sample sheets for the instruments, exporting billing information, and tracking completion of orders.
The Vermont Genetics Network (VGN) Outreach Program links the core facilities at UVM to colleges around the State of Vermont. Our outreach programs inspire Vermont undergraduates within and outside our Baccalaureate Partner Institutions by providing visits from VGN staff and faculty, who work closely with students and college faculty to implement cutting-edge experiments in their course settings.
We have been delivering the microarray module to students throughout the state since 2003. This program has led to numerous collaborations between faculty at Vermont baccalaureate colleges and the DNA Analysis and Microarray Cores at UVM. One of the goals of microarray outreach is the integration of this technology into the science curriculum at colleges throughout Vermont. We are seeing this goal being achieved with subsequent deliveries at several colleges, including Johnson State College in the Spring of 2006, St. Michael’s College and Middlebury College in the Spring of 2007, and Norwich University and Green Mountain College scheduled for the fall of 2007.
Two subsequent outreach modules are being developed. The first is the bioinformatics outreach tutorial. This tutorial was developed in the spring and summer of 2006 in association with Dr. William Barnes from Clarion University of Pennsylvania. The beta test of the module was run at the University of Vermont in the fall of 2006 and is scheduled for its first outreach delivery at Green Mountain College in the fall of 2007. The second is a proteomics module currently in the developmental stages and anticipated to be beta tested in the fall of 2008.
All of the VGN outreach activities invest in the level of biomedical research throughout the State of Vermont. The outreach modules and the networking that is achieved through these modules help to bring about sustainable changes in how we in Vermont carry out research and educate our next generation of scientists.
The Core Technologies (CT) Unit, located at the Eastern Regional Research Center (ERRC), is a centralized resource of advanced instrumentation, technologies, and multidisciplinary expertise. Its mission is to provide cutting-edge complementary research data and informational input support for a broad range of research programs approved by the Agricultural Research Service (ARS), the in-house research arm of the United States Department of Agriculture. The CT Unit is comprised of four research-related components: genetic analysis, proteomics-biopolymers mass spectrometry, microscopic imaging, and magnetic resonance spectroscopy (NMR). In addition, the Scientific Information Resources (Library) and the Research Data System operations of CT provide the means to facilitate data dissemination to stakeholders and the general public through up-to-date information techniques. This coordinated organization assures dependable and quality support to the Agricultural Research Service community.
The major change in our core facility has been the installation of the Illumina 1G high-throughput sequencing instrument. We continue to improve both our computational infrastructure, to accommodate data throughput, and our Web ordering system. Services available for fee and multi-user instruments available for researchers to use independently are also included on the poster.
Pennington Biomedical Research Center is the world’s largest academic research center focused on nutrition research. Pennington has 80 faculty members and 600 physicians, scientists, and support staff. The Genomics Core Facility serves the basic sciences research program. It has been singled out from nineteen Pennington core laboratories as the standard to which the other core laboratories should aspire. The Genomics Core Facility occupies a state-of-the-art core laboratory plus two smaller ancillary laboratories. It provides DNA sequencing; DNA fragment analysis; qualitative and quantitative analysis of DNA, protein, and RNA samples; quantitative PCR; microarray RNA labeling, spotting, hybridization, and scanning; robotics, automated DNA and RNA extraction; and bioinformatics services. Individual and small group training and consultation services are offered for sequence analysis, microarray analysis, primer design, and real-time PCR. The laboratory provides services to laboratories outside of Pennington Biomedical Research Center, including Louisiana State University main campus and School of Veterinary Medicine, Southern University, Southeastern Louisiana University, Tulane, and LSU Medical School. It serves over 300 researchers on an annual basis.
The Biopolymers Facility at Harvard Medical School is a service center providing a host of genomics services to the Longwood Medical Area community. Technologies offered include DNA sequencing, genotyping, Affymetrix gene chips, next generation DNA sequencing (Illumina Genome Analyzer), oligonucleotide ordering, and reagents and supplies ordering. The facility has a comprehensive Web-based laboratory information management system (LIMS), which allows our users to order services and supplies, track experiment progress, retrieve data, and review and pay invoices.
The Stowers Institute Molecular Biology core facility utilizes robotics instruments to provide high-throughput services as well as to complete custom collaborative projects. The primary instrumentation of our core facility consists of two Biomek FX liquid-handling instruments, a Qpix2 colony picking/manipulation robot, a Singer RoTor HDA, a Hydra, and a Labchip 90. Protocols for assays have been developed on these instruments to support and further the research of the labs within our institute. Services supported by these instruments include sequencing setup/ cleanup, 96-well plasmid preps, and 96-well RNA isolations. Custom project examples demonstrated here include 96-well bacterial/yeast transformations (plating and picking), microtiter plate serial dilutions and pinning in 384 format viability assays, and synthetic genetic arrays (SGA) and complex haploinsufficiency assays in 1536 format.
The Core Facility (Core Laboratory, CL) at Masaryk University was originally founded to support on-campus research; today, CL services cover not only the campus area, but other areas in the country and abroad as well. The CL takes part in several biomedicine studies, participates in teaching courses, and offers hands-on courses and consultation services. The CL is an active member of the Association of Biomolecular Resource Facilities (ABRF).
DNA sequencing. Automatic DNA sequencing based on capillary electrophoresis with laser detection. Plasmids and PCR products sequencing. Identification of point mutations, insertions, and deletions in the sequence.
DNA fragmentation analysis. Microsatellite analysis, genotyping for genetic linkage studies, paternity identification, AFLP fingerprinting, and mutation detection.
Oligonucleotide synthesis. Synthesis of DNA, RNA, modified oligonucleotides (fluorescent labels, double-labeled real-time PCR probes, biotinylated, phosphorylated, degenerated oligos, etc.), phosphorothioates. Oligonucleotide purifications based on the planned application.
Current projects cover proteomes of different organisms (bacteriophages, bacteria, yeast, plants, etc.), including a search for important diagnostic biomarkers in human plasma and various types of human cells.
Sample preparation. Optimization of conditions for solubilization, prefractionation before protein separation, depletion of abundant proteins, specific fractionation/ enrichment techniques to separate groups of proteins (peptides), purification of recombinant proteins, etc.
Protein separation. Electromigration methods - isoelecric focusing in a polyacrylamide gel with an immobilized pH gradient, polyacrylamide gel electrophoresis (1-DE, 2-DE), various types of staining, image analysis.
Chromatographic methods—various types of peptide and protein separations, multidimensional separations, and LC-MALDI applications.
Protein characterization. Intact protein analysis, peptide profiling, characterization of microorganisms (MALDI-MS/MS), protein identification by peptide mass fingerprinting and MS/MS methods, assigning of site and type of post-translation modification (e.g., phosphorylation). MALDI-MS/MS and LC-MS/MS instrumentation.
Contact at http://www.sci.muni.cz/FGP
Supported by grant projects MSM 0021622415 and LC 06034.
Nemours is the nation’s largest medical group practice devoted to pediatric care, education, and research. It is the mission of Nemours to provide leadership, institutions, and services to improve the health of children. As a part of that mission, Nemours’ Biomedical Research is committed to scholarly and scientific endeavors directed towards improving the diagnosis and treatment of pediatric medical conditions. With locations in Wilmington, DE, as well as Jacksonville, Orlando, and Pensacola, FL, more than 40 different research programs and laboratories support the medical and surgical staff in restoring and improving the health of acutely and chronically ill children. Nemours has invested in patient-oriented and science-based research and in services that bridge the gap between bench and bedside. Nemours supports CLIA-certified facilities performing diagnostic tests for pediatric genetic diseases (www.genetests.org), immunological and gastrointestinal disorders, solid organ transplant, infant pulmonary assessment, speech and hearing assessment for craniofacial disorders, and histochemistry tests for neuromuscular diseases. Nemours’ Biomedical Research has also invested in research service cores whose principal mission is to support translational research programs that move discoveries rapidly from bench to bedside and to the medical community. The Nemours’ research cores offer services to research and clinical labs using molecular genetics, histotechnology, bio-informatics, mass spectrometry, cell culture, proteomics, and flow cytometry. Nemours’ Biomedical Research continued support enabled the establishment of a new Center for Pediatric Research that is funded by a grant from the National Center for Research Resources, under the Institutional Development Award Program of the National Institutes of Health.
The mission of the Center for Nutrition Research Unit Mass Spectrometry Core Facility at the University of Colorado Denver is to provide investigators with the means to identify, characterize, and quantify molecules of interest in cells, tissues, and biofluids. Our well-equipped laboratories provide analytical services and participate in the development of new assays and technologies in the areas of mass spectrometry, proteomics, metabolomics, pharmacokinetics, and isotope labeling studies. In addition to investigators affiliated with the Human Nutrition Center, we provide services to other groups within the University of Colorado Denver, including the Schools of Medicine and Pharmacy, graduate students and researchers at our Downtown Denver Campus, University Hospital, and Children’s Hospital. We also offer our services to outside and industrial partners.
Our analyses include two-dimensional gel and multidimensional chromatography workflows for proteomics; quantitative measurements of drugs and hormones; biochemical profiling of metabolites; gas isotope ratio determinations for energy studies (total body water, total energy expenditure); as well as pharmacokinetic and clinical studies. We offer expert assistance throughout the analytical workflow, from sample preparation to bioinformatic analysis. We also provide comprehensive instruction for interested investigators, have developed a hands-on proteomics and metabolomics educational program, and are extending our curriculum to pharmacokinetics and other aspects of systems biology.
Our equipment consists of two quadrupole ion traps equipped with nanospray, HPLC-Chip and AP-MALDI sources, one electrospray time-of-flight mass spectrometer, three triple quadrupole mass spectrometers, an isotope ratio mass spectrometer, a GC-MS instrument, a fluorescence imager, and a preparative scale HPLC system. We also have access to 4000 QTRAP and TOF-TOF instrumentation, allowing investigators to adopt multiple strategies for results validation. In addition to collaborative research, our scientists conduct their own studies in the areas of immunosuppression, oxidative stress, diabetes, and obesity. For more information, please contact Karen Jonscher at karen. ude.cshcu@rehcsnoj.
The BioMedical Genomics Center (BMGC) at the University of Minnesota is a dynamic core currently consisting of five distinct facilities that provide comprehensive, cost-efficient, and cutting-edge instrumentation, infrastructure, and technical expertise for genomics research to the university as well as the extended local scientific community. BMGC facilitates almost every aspect of genomics research, including structural genomics, functional genomics, and comparative genomics, through its core facilities, which include:
Each of our facilities is staffed and maintained by a group of highly experienced academic and technical members. They are not only specialists in their respective areas of expertise but also cross-trained in operation of multiple instruments to provide more comprehensive technical support.
We aspire to be a regional center for genomics research by providing researchers easy access to state-of-the-art technology and techniques available in the rapidly advancing fields of genomics. By sharing of technology and expertise and applying the vast amounts of information provided by sequencing of human, animal, and microbial genomes, we hope to contribute significantly in the identification and development of better ways to treat and prevent disease.
The mission of the Proteomics and Metabolomics Core (PMC) is to serve as an enabling resource for research and development programs at Colorado State University. We strive to build instrumental capabilities that exceed the normal resources of individual research programs, and make those technologies available as a shared resource. We also aim to provide an environment rich in expertise and educational resources, and to foster collaboration across the CSU community and beyond.
The primary focus of the PMC is mass spectrometry–based proteomics and metabolomics. The PMC currently houses a wide variety of mass spectrometers, including two ion traps, a MALDI TOF-TOF, and a Q-TOF. Proteomics capabilities range from simple protein identifications to the characterization of post-translational modifications and the analysis of complex mixtures. In addition, we support quantitative proteomics applications utilizing various labeling techniques, such as ITRAQ and DIGE. Our metabolomics program is primarily focused on discovery-based LC-MS profiling and biomarker discovery. We utilize UPLC chromatography and Q-TOF mass spectrometry in combination with multivariate statistical data analysis. The PMC also operates an enzyme freezer program, which provides in-stock availability of many commonly used reagents and supplies for the CSU community, as well as routine DNA sequencing.
The Molecular Biology Core facility at Stowers Institute for Medical Research is a cutting-edge laboratory dedicated exclusively to supporting internal research efforts. We perform a number of routine services and continually strive to obtain specialized expertise to act as a teaching resource. Our routine services include sequencing, genotyping, site-directed mutagenesis, riboprobe synthesis, clone ordering and banking, pathogen testing, high-throughput DNA and RNA extractions, and robotics. In addition, our efforts to collaborate with Institute labs and stay informed of new technologies allow the Molecular Biology Core to have a large impact on scope and potentiality of research projects.
The University of Utah DNA Sequencing and Genomics Core Facility offers a variety of genetic analysis services to both on-campus and off-campus researchers. The details of these services and the workflow associated with them will be discussed. The sequencing portion of our lab provides DNA sequencing using an Applied Biosystems 3730xl instrument. A standardized sample submission process increases workflow efficiency, and Geospiza’s Web-based system allows users to securely view their sequencing data online. In addition to standard DNA sequencing, our facility offers a mutation detection service to identify single-nucleotide polymorphisms (SNPs), insertions, and deletions. Researchers select genes or regions of interest and our facility designs and optimizes the PCR primers, performs the initial PCR, runs the sequencing reactions, and analyzes the data using SoftGenetics Mutation Surveyor software. The genomics portion of our lab provides full-service genotyping from PCR setup through analysis. Our facility has commercial and custom sets of fluorescently labeled microsatellite markers that can be used for whole-genome linkage studies and fine-mapping projects. Particular sets of markers can be used for loss of heterozygosity and micro-satellite instability studies. An Applied Biosystems 3130xl instrument is used to run genotyping samples, and Applied Biosystems GeneMapper software is used for data analysis. The 3130xl instrument is also available for researchers with fluorescently labeled PCR products that are ready to run. SNP genotyping is available using Applied Biosystems Taqman genotyping assays, and our facility will incorporate a high-throughput SNP genotyping system in the near future. Two Applied Biosystems 7900HT instruments are available for researchers interested in performing real-time PCR experiments for gene expression studies. A Biomek FX and a Velocity 11 VPrep robot are available to researchers who require automated pipetting for their experiments.
There seems to be a perception that the small core facility is disappearing in the age of systems biology. The Molecular Biology Core Facility (MBCF) at the Trudeau Institute is alive and well, providing a variety of services to the investigators at the institute while recovering the majority of its expenses. The MBCF survives by offering an assortment of services that are specifically tailored to the institute mission and by personalizing each service to the individual investigator. A five-year core grant, as part of a program project, helps to cover the cost of developing new techniques. Services provided include DNA sequencing; spectratyping of the T-cell repertoire (fragment analysis); production, purification and labeling of major histocompatibility (MHC) Class I and Class II multimers for fluorescent activated cell sorter analysis; real-time PCR measurement of gene expression and viral loads; knock-out mouse and Mycoplasma screening; DNA haptenation and recombinant protein expression/purification for antibody detection and in vivo immunization. A variety of simple services such as primer design, primer ordering, stock primers, peptide ordering, and Taq production save the investigators time, effort, and money. Education and training are provided for all techniques and for using MBCF instrumentation, such as spectrophotometers, real-time PCR equipment, and image capture/analysis equipment. Not only does the MBCF provide the service, the personnel assist in the planning and analysis to maximize proper usage of the technique. The MBCF at Trudeau Institute prospers through versatility and customization.
The UC Davis Genome Center (GC) opened three years ago in a state-of-the-art facility and combines bench science with computational approaches to investigate questions in genomics. An important part of the GC is its five technology service cores, providing access to enabling technologies to researchers within and outside the UC Davis campus. The DNA Technologies Core provides custom high-throughput SNP genotyping using the Illumina GoldenGate assay. In addition to analysis of human and murine SNPs, we have used the Illumina BeadArray system for genotyping diverse animal and plant species; these include loblolly pine, lettuce, cotton, and cat, as well as genome-specific genotyping of allopolyploid wheat populations and radiation hybrid mapping of armadillo. Recent acquisition of the Illumina BeadXpress genotyping platform expands our capabilities to cost-effectively genotype from one to several thousand SNPs in varying numbers of individuals. In addition to offering Illumina array-based gene expression services, the Expression Analysis Core specializes in chromatin immunoprecipitation (ChIP) techniques. These methodologies are generally unavailable in technology service cores. We offer hands-on workshops in the basic manipulations required for chromatin immunoprecipitations as well as carrying out ChIP as a service with user-provided cells and antibodies. Additionally, our ability to process and analyze NimbleGen arrays allows the core to carry out complete ChIP-chip experiments. Both cores share use of an Illumina Genome Analyzer for ultra-high-throughput sequencing, allowing further services to be provided by both core facilities. These include SNP discovery, re-sequencing, ChIP-seq, and non-array-based expression profiling. More information on these and the other GC cores is available at http://www.genomecenter.ucdavis.edu/corefacilities.html.
The Cornell University Life Sciences Core Laboratories Center (CLC) provides an array of genomics, proteomics, imaging, and informatics shared research resources and services to the university community and to outside investigators. With a concentration of advanced instrumentation and expertise in their applications, the CLC is a key resource for life-sciences basic research and medical research investigators at Cornell University and at other academic institutions and commercial enterprises.
The DNA Sequencing and Genotyping Facility of the Cornell University Life Sciences Core Laboratories Center provides an array of shared research resources and services to the university community and to outside investigators. The facility provides a concentration of advanced instrumentation and expertise in their applications. These services include several options for Sanger sequencing of plasmid and PCR products, as well as an Illumina Genome Analyzer for massively parallel sequencing projects. Also, this facility provides SNP genotyping using automated sample-processing pipelines for the Applied Biosystem SNPlex technology, Illumina GoldenGate and Infinium technologies, and Affymetrix SNP 5.0/6.0 and Targeted Genotyping projects. The goal of the facility is to meet the increasing need of Cornell investigators for rapid and accurate DNA sequencing and genotyping.
The Functional Genomics Center Zurich (FGCZ) is a joint state-of-the-art research facility of the University of Zurich and the Swiss Federal Institute of Technology (ETH) Zurich. With the latest technologies and expert support in genomics, transcriptomics, proteomics, metabolomics, and bioinformatics, the FGCZ carries out research projects and technology development in collaboration with the Zurich Life Science research community and offers services to other academic and commercial scientists via three different modes:
The analysis of underivatized lipids within body fluids as well as cell and tissue extracts is still a very challenging task with great biochemical and clinical relevance. Anomalous lipid concentrations are correlated to neoplastic and neurodegenerative diseases, diabetes, etc. Structural diversity of each lipid or lipid class respectively will have a distinct effect on membrane properties (fluidity, permeability, oxygen scavenger, etc.).
Therefore, the advantage of recombined use of HPLC, MS, and NMR will be shown. Many studies have dealt with the analysis of lipids, but to our knowledge nobody has used a combinatorial approach so far. A combination of HPLC separation power, MS sensitivity with accurate mass measurement of molecular and fragment ions, and NMR structure elucidation power will meet the challenge. The low NMR sensitivity can be compensated by preceding concentration steps via HPLC and fraction sampling. New HPLC methods for several phospholipid classes (sphingomyeline, phosphatidylcholine, phosphatidylenthanolamine) were developed, and the retention times and the detected masses were determined. Location of fatty acids with respect to position sn-1 and sn-2 were identified in negative ion mode by the relative intensity of their [M-H] ions and the neutral loss of the fatty acid ketene. In positive ion mode, the polar head group was cleaved off. The molecular formula was generated by matching high mass accuracy and isotopomer pattern. The separated fractions were assigned by means of the 1D- and 2D-NMR spectra. Saturated, mono-unsaturated (MOFA), or polyunsaturated fatty acids (PUFA) show zero, two, or four carbon signals between 120 and 130 ppm. The MOFA and PUFA reveal unambiguously different chemical shifts for the olefinic carbons. However, lipids with MOFAs have similar olefinic carbon shifts. Nonetheless, a lipid with two MOFAs is deduced from the intensity ratio of the olefinic protons with respect to the glycerol protons.
BAC fingerprinting provides an efficient and cost-effective method of characterizing large genomic fragment libraries for genome sequencing, positional cloning, and physical mapping efforts. Here, we present details about the application of a new high-density size standard to accurately size large DNA fragments generated by BAC fingerprinting. The method described facilitates DNA fragment sizing up to 1200 bp and exhibits a high degree of precision and robustness in a high-throughput environment.
Reduced bone loss was observed in femur by dual-energy X-ray absorptiometry from monocyte chemotactic protein 1–deficient (MCP-1-/-) mice relative to that from wild-type mice. Absence of MCP-1 reduced bone loss from ovariectomy. MCP-1 is a CC chemokine found at the site of bone loss related to inflammation. After stimulation by receptor activator of NFκB ligand (RANKL), the expression level of MCP-1 is increased in the bone marrow–derived macrophages (BMM). Blocking of MCP-1 inhibited osteoclast formation, and exogenously added MCP-1 stimulated it in RANKL-stimulated BMM. We determined osteoclast formation, using MCP-1 deficient mice in the background of C57BL/6 to investigate the role of MCP-1 in bone metabolism. However, no difference was observed in osteoclastogenesis evaluated by tartrate-resistant acid phosphatase activity. Bone loss could be not due to MCP-I induced by RANKL during osteoclast differentiation.
This work was supported by SRC funding to IRC, University of Ulsan, from KOSEF and Ministry of Korea Sciences and Technology, and by KOSEF grant funded by the Korea government (MOST) (R01200700021 08202007).
Treatment of retroviruses with tetraethylthiuram disulfide (Antabuse) results in chemical inactivation of retroviral infectivity while maintaining functional envelope glycoproteins. We characterized the inactivated virions.
We used microscale HPLC methods, coupled with SDS-PAGE, immunoblot analysis, mass spectrometry, and protein sequence analysis of eluted proteins to characterize Antabuse-modified viral proteins.
We biochemically characterized the targets and mechanisms of inactivation involved in tetraethylthiuram disulfide treatment of virions, demonstrating that gag proteins (CA, MA, NC) and the enzymes RT, PR, and IN can be covalently modified by tetraethylthiuram disulfide treatment. In vitro studies in cell-free systems and in cell culture indicate that, as judged by both biochemical and biological (infectivity) criteria, inactivation by tetraethylthiuram disulfide does not appear to be reversible.
Tetraethylthiuram disulfide treatment results in effectively irreversible inactivation of infectivity through covalent modification of key viral proteins, including NC. The inactivation procedure preserves the conformational and functional integrity of the viral envelope glycoproteins. Contract No. N01-CO-12400.
The routine handling of RNA for gene expression studies often requires one or more freeze-thaw events prior to downstream synthesis reactions. Because this is common procedure with many RNAs and cDNAs used for gene expression studies in microarray and real-time quantitative polymerase chain reaction (RT-qPCR), it is of interest to study the affects of freeze-thaw cycling. These studies were designed to mimic the regular handling procedures employed on a daily basis in many labs.
In this study, total RNA isolated from rat brain tissue in RNase-free water and cDNA prepared from HeLa cell RNA and suspended in first strand reverse transcriptase buffer, DTT, dNTP, and Superscript III, were frozen and thawed from –20°C to room temperature up to ten times. Following treatment, samples were analyzed using the Agilent Bioanalyzer 2100, Affymetrix microarray rat GeneChip 230A, and an Applied Biosystems HPRT assay on demand for RT-qPCR. Results from the microarray study on total RNA indicate little to no effect on gene signatures, suggesting that the ability of RNA to withstand repetitive freeze-thaw episodes is remarkable. Not surprising, the RT-qPCR results for the cDNA revealed very small but noteworthy decreases in gene detection, indicating that “uncleaned” cDNA is slightly affected by freeze-thaw cycling. It should be noted, however, that these effects were nearly insignificant and are highly dependent on individual handling techniques, sample purity, and each lab’s handling and storage protocols.
The 454 Life Sciences Genome Sequencer FLX (GS-FLX), successor of the GS-20, is a high-throughput DNA sequencing system. Its applications include whole-genome sequencing, small RNA sequencing, transcriptional analysis, and ultra-deep sequencing of target genes. 454 technologies allow whole-genome sequencing and resequencing in significantly less time and at lower cost than traditional Sanger sequencing. 454 is the first advanced sequencing technology that generates hundreds of thousands of DNA sequences in one run. It has a simple, unbiased sample preparation and high capacity for massively parallel sequencing. It has dramatically increased the feasibility and lowered the cost of large-scale DNA sequencing projects.
Briefly, the process includes ssDNA library preparation and amplification followed by a sequencing-by-synthesis method. Library preparation includes a step to determine the optimal quantity of input DNA that will result in a single effective copy/bead for the amplification procedure. This is done by carrying out emPCR reactions with a titration of the library and identifying the amount of input DNA that provides the best sequencing result when the clonally amplified and bead-bound products are sequenced using the GS-FLX system. Although this procedure worked with many samples on GS-20, it did not always give us the expected results on the GS-FLX. The titration step also gives an additional expense to the researchers. Here, we report a method that eliminates or minimizes titration and, therefore, decreases the total cost of 454 sequencing.
Interleukin-6 (IL-6), a cytokine secreted by monocytes/macrophages and other cells, induces the secretion of acute phase proteins, specifically, C-reactive protein (CRP), by hepatocytes. Although CRP is involved in innate immunity and participates in microbial killing by neutralizing toxins released by phagocytes following tissue injury and/or infection, elevated levels are a risk factor for cardiovascular disease (CVD) and metabolic syndrome. IL-6 secretion is induced by infection, lipopolysaccharides (LPS), IL-1, and TNF-α. Mushrooms have been shown to protect from infections due to their immunomodulatory effects. Previously, we reported that white button mushroom (WBM) extracts increased the secretion of TNF-α by THP1 cells (EB07). Their effect on IL-6 secretion has not been previously investigated. Considering the role of TNF-α on IL-6 secretion, we hypothesized that WBM extracts would increase IL-6 secretion. To test our hypothesis, we incubated THP1 cells, 500,000/mL, with 0, 0.1, 1, and 10 μg/mL ± LPS (2.5 and 10 μg/mL) in serum-free transferrin-selenium-insulin-supplemented medium for 48 h. IL-6 was measured in the supernatant by ELISA. In contrast to what we expected, in the absence of LPS, WBM extracts decreased IL-6 levels by 42% (mean ± SEM, pg/mL, n = 4–6: 24 ± 3 versus 13.6 ± 3.33; p < 0.05, ANOVA). IL-6 decrease was not due to apoptosis as assessed by trypan blue exclusion test, but very likely to gene regulation. In LPS-treated cells, WBM extracts increased IL-6 levels by 10%–34% (369 ± 22 versus 494 ± 30; p < 0.05). Regardless of WBM extracts dose, LPS increased IL-6 secretion by 15- to 36-fold (p < 0.001). Our data suggest that in a non-infected individual, WBM may decrease CRP secretion and very likely CVD risk. Down-regulation of IL-6 would not compromise the response to infection because of up-regulation of α-defensins 1–3 by WBM (Kuvibidila and Korlagunta, EB07). In vivo work is underway to confirm our observation. Support: Grant #580790706 USDA/Mushroom Council/NutriCore and Oklahoma State University.
We present details about developing methods to take advantage of a new high-performance, 68 peak size standard and illustrate its versatility, accuracy, flexibility, and precision when used on various capillary electrophoresis instrument platforms, with different polymers and capillary array lengths. Methods described include DNA fragment sizing up to 1200 bp in applications such as AFLP, T-RFLP, VNTR, mutation screening, MLST, and BAC fingerprinting.
SeqWright, Incorporated, is a molecular biology research support company established to offer comprehensive and cost-effective solutions to life science researchers worldwide. SeqWright gives its customers access to world-class expertise and experience in many diverse areas, including, but not limited to: DNA sequencing, SOLiD sequencing, SNP identification and discovery, whole-genome amplification, shotgun library construction, vector construction and mutagenesis, quantitative PCR from DNA or RNA, microbial identification testing, and genotyping. SeqWright is also an Affymetrix-authorized service provider of micro-array expression analysis and SNP-genotyping services. In addition, SeqWright offers an innovative, whole-genome methylation scan service, developed by SeqWright for use on the Affymetrix platform. Our services have been used in the completion of a number of major collaborations and endeavors, including the Human Genome Project, as well as in ongoing clinical trials. Our strong reputation, which has led to preferred and/or sole-supplier status with several of the world’s largest pharmaceutical companies, was built on a thirteen-year track record of quality results and technical expertise. SeqWright employs a LIM system, the Finch Suite Server Database, which enables sample tracking from beginning to end throughout our production pipeline. SeqWright is CLIA certified and adheres to 21 CFR Part 58, Good Laboratory Practice (GLP) for Non-Clinical Laboratory Studies, as outlined in the Code of Federal Regulations (CFR) by the Food and Drug Administration (FDA). With a commitment to quality and technological advancement, SeqWright is positioned to remain at the forefront of contract genomic research for years to come.
The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection and analysis significantly improves the analytical results in terms of lower background, higher number of detected phosphorylation sites, and enhanced system sensitivity.
Recently, there has been an increase in offline chromatographic approaches regarding selective enrichment of phosphorylated peptides using metal oxide chromatography with titanium dioxide and zirconium dioxide media. In this paper, we have tested various combinations of loading and washing conditions, column geometry, and materials in order to improve the performance of the method used in our laboratory. The tests included the investigation of the impact of different additives to loading and washing mobile phases. The experiments described in this publication were performed using a mix of synthetic peptides (phosphorylated and non-phosphorylated) with an equal amount of tryptically digested bovine serum albumin in order to mimic a complex sample environment and to test the level of binding of unphosphorylated peptides. To test the best method resulting from the optimization procedure, tryptic digests of protein complexes were treated on TiO2 or ZrO2 stationary phases. After enrichment, it was possible to identify 152 phosphorylation sites from four protein complexes, including proteins not visible on a silver-stained SDS-PAGE gel. In summary, our improved method is highly effective for the enrichment of phosphopeptides from biological samples prior to mass spectrometry, and is suitable for high-throughput phosphoproteomic projects that aim to uncover phosphorylation-dependent signaling pathways.
Peptides with one phosphate group cannot be cleanly separated from peptides with no phosphate by anion-exchange chromatography, since the electrostatic repulsion of the positively charged termini outweighs the electrostatic attraction of the phosphate group to the stationary phase. When the column is operated in the hydrophilic interaction chromatography mode, though, the hydrophilicity of the phosphate group plus its electrostatic attraction accomplishes this separation despite the electrostatic repulsion of the termini. This combination is called electrostatic repulsion–hydrophilic interaction chromatography (ERLIC). Selectivity for phosphate groups is ensured by operation at pH 2. A gradient to 0.2 M triethylamine phosphate elutes peptides with one to four phosphate groups. Retention is much greater using volatile salts such as ammonium formate, to the point that it is practical to isolate phosphopeptides via solid-phase extraction in the ERLIC mode. Unlike high-affinity methods involving titania or immobilized metal affinity chromatography, ERLIC is sensitive to aspects of peptide composition besides the phosphate group. This makes it suitable as a high-resolution mode for samples containing thousands of phosphopeptides.
The emergence of protein methylation in transcriptional regulation and cellular signaling exemplifies the importance of this small post-translational modification in cellular biology. The most publicized role of protein methylation is in histone tails. In this process, lysine and arginine residues are modified, resulting in the alteration of the transcriptional state of associated DNA. Methylation has also been found in numerous other proteins, including those involved in translation. In this study, mass spectrometry has been used to identify the location, type, and in several cases the enzyme responsible for the methylation of specific proteins of the baker’s yeast large ribosome subunit.
The yeast ribosome provides an ideal complex to investigate protein modification using the recently developed top-down proteomics techniques on the Thermo Finnegan LTQ-FT hybrid mass spectrometer. Bioinformatic methodologies were used to search the Saccharomyces cerevisiae genome, and new hypothetical protein methyltransferases were found. Ribosomal proteins from yeast strains lacking these genes were isolated and screened for loss of methylation using LC-MS on a SCIEX API III+ mass spectrometer. When loss of methylation was detected, LC fractions were collected and analyzed using either traditional bottom-up proteomics (enzymatic digestion followed by MS/MS of the resulting peptides) or, more recently, using a top-down approach fragmenting the intact protein.
The use of bioinforamtics, the simplicity of yeast genetics, and the power of mass spectrometry have led to the discovery of multiple new methyltranseferases, their associated ribosomal substrates, and unique unexpected protein modifications of the ribosome.
Proteins are extensively modified after translation due to cellular regulation, signal transduction, or chemical damage. Peptide tandem mass spectrometry can discover post-translational modifications, as well as sequence polymorphisms. Recent efforts have studied modifications at the proteomic scale. In this context, it becomes crucial to assess the accuracy of modification discovery. We discuss methods to quantify the false-discovery rate from a search and demonstrate how several features can be used to distinguish valid modifications from search artifacts. We present a tool, PTMFinder, which implements these methods. We summarize the corpus of post-translational modifications identified on large datasets. Thousands of known and novel modification sites are identified, including site-specific modifications conserved over vast evolutionary distances.
A complete analysis of the phosphoproteome is critical for the understanding of the control of cellular metabolism by protein kinases (phosphorylation) and protein phosphotases (dephosphorylation). Drawbacks in the analysis of phosphorylation and dephosphorylation are that phosphorylated signal proteins are of rather low abundance in cells. Mass spectrometry of phosphopeptides has become a powerful tool for phosphorylation site identification. However, there is a general need for specific efficient enrichment strategies of phosphorylated peptides to compensate for the rather low abundance of phosphopeptides. Titanium dioxide and immobilized metal ion affinity chromatography, alone or in combination with other methods, enrich phosphorylated polypeptides without bias towards a specific phosphorylated amino acid. In this context, we developed and optimized specific enrichment protocols based on proprietary materials, combined with pre-treatment steps to decrease acidic peptides that cause high background. Using these optimized methods, we successfully enriched phosphopeptides from digests of complex biological samples, including tissues and cell lines. The specificity, efficiency, sensitivity, and robustness of these methods have been analyzed using both ESI and MALDI-MS.
Post-translational modification of proteins plays a fundamental role in cellular processes, and their determination is one of the main goals of modern proteomic research. Phosphorylation, glycosylation, and acylation are the best characterized; however, the variety, diversity, and heterogeneity of these modifications requires novel analytical tools for their qualitative and quantitative assessment. We have investigated the potential of a novel traveling-wave ion mobility spectrometer equipped with both MALDI and atmospheric ionization sources for the separation, detection, and mass determination of post-translationally modified proteins. Species were ionized and the resulting ions separated based upon their ion mobility, or collision cross section, through the ion mobility spectrometer (IMS), and subsequently mass analyzed using the oa-TOF analyzer.
Peptides and phosphopeptides originating from proteolytic digestion of the protein mixture were analyzed directly or purified using TiO2 columns as previously described. The LC-IMS-MS separations were performed using a nanoscale UPLC system in trapping mode (described previously), in combination with a Synapt HDMS instrument. In these experiments the m/z, drift time, and UPLC retention time of phosphorylated and non-phosphorylated species was determined. We will show the potential of IMS in combination with mass spectrometry to discriminate between these species and show that by synchronizing the output from the IMS device with the pusher pulse of the oa-TOF mass analyzer, we improve sensitivity 3- to 10-fold compared to the normal TOF MS mode of acquisition.
In addition, by raising the collision energy on the traveling wave, collision cell fragmentation of the peptide or modified peptides can be induced, allowing structural information to be obtained. We will present data obtained from glycopeptides analyzed by MALDI-IMS-MS. The released glycans were initially analyzed using IMS-MS; then further analysis was performed on the individual glycans using time aligned parallel (TAP) fragmentation.
Glycosylation plays key roles in controlling various biological processes. It is one of the most widespread and complex post-translational modifications (PTMs) found on proteins, and its characterization remains a great analytical challenge. LC MS/MS is the most powerful and versatile technique for glycopeptide structure elucidation. However, commonly used collisional-induced dissociation (CID) has limitations on determining the modification site, due to the labile nature of the glycan modifications. As a new dissociation technique, electron transfer dissociation (ETD) preserves labile PTMs and provides a new and powerful tool that makes the identification of modification sites possible. Since glycosylated proteins and the resulting peptides are most often highly heterogeneous, high-quality liquid chromatography is critical for glycopeptides analysis. In this study, two reasonably well characterized glycoproteins, bovine α1-acid glycoprotein and human α1-acid glycoprotein were analyzed using nano-LC MS/MS with ETD. Liquid chromatography separation conditions were systematically optimized for glycopeptides analysis prior to mass spectrometry using a graphitic carbon column. Unlike traditional reversed-phase column, the interaction between the graphite column and analyte is very dependent on the molecular area, the type and positioning of the functional groups at the points of contact. The graphitic carbon column (Hypercarb, Thermo Scientific) demonstrated excellent capabilities for glycopeptides analysis, especially for short hydrophilic peptides containing bi- or tri-antennary glycan chains without any enrichment. Formation of metal adducts on the Hypercarb column promotes higher charge species, and as a result promotes ETD fragmentation of glycopeptides. The combination of porous graphite chromatography and ETD-MS/MS is demonstrated to be a useful and flexible tool for studying glycosylation and identifying PTM sites.
The DNA damage response pathway is critical in maintaining genome stability, and proteins within this pathway are commonly mis-regulated in cancer cells. Camptothecin is an anti-cancer drug that inhibits topoisomerase I DNA unwinding and leads to DNA damage in cells undergoing DNA replication. Here, we employed a mass spectrometry (MS)-based proteomics approach to identify and characterize campothecin-induced DNA damage response proteins in A549 cells. A549 cells were treated with 5 μM camptothecin and harvested at 0, 2, 8, and 24 h post treatment. Phosphoproteins were enriched using phosphoprotein metal affinity chromatography (PMAC). Multiplexed iTRAQ labeling was used to determine relative changes in overall protein abundance and phosphorylation state of proteins. The labeled samples were analyzed using nano-LC-ESI-MS/MS with LTQ Orbitrap XL. Selected parent peptide ions were fragmented in the high-energy collision dissociation (HCD) cell. The HCD MS/MS spectra have high mass accuracy and resolution since they are acquired in the orbitrap and display similar fragmentation patterns to those from a quadrupole collision cell. More than 500 proteins were quantified by iTRAQ (CV < 15%) using the HCD method from A549 whole cell and PMAC lysates. Phosphoprotein enrichment by PMAC resin increased sample diversity by 145 additional proteins identified. To specifically quantify the phospho sites, we further purified phosphopeptides from the iTRAQ PMAC fraction by IMAC (gallium-IDA) spin columns. After IMAC enrichment, we were able to identify and quantify an additional 30 phosphorylation sites from a very limited amount of sample (15 μg of PMAC fraction).
Two novel techniques have recently been developed that can be applied to the analysis of protein phosphorylation. Here we seek to better understand the utility of multi-stage activation (MSA) and electron transfer dissociation (ETD) applied to the analysis of sites of phosphorylation.
Enzymatic digests of recombinant human kinases were analyzed by LC-MS performing NLMS3, MSA, or ETD. The ETD was performed on a linear ion trap mass spectrometer whereas the NLMS3 and MSA were performed on either a linear ion trap or a hybrid linear ion trap electrostatic orbital trapping mass spectrometer. Data were analyzed manually and using Mascot and SEQUEST database search algorithms against a proprietary database of human kinases and against the SwissProt database (human entries).
The different dissociation techniques were found to be complementary. Typically, peptides carrying three or more charges produced high-quality data by ETD, whereas peptides carrying two or three charges worked well by MSA and NLMS3. In addition, peptides bearing multiple sites of phosphorylation tended to produce poorer quality data by the neutral loss-based techniques, whereas this was less likely by ETD. Lastly and previously known, peptides containing phosphotyrosine did not produce a significant neutral loss and so did not initiate the acquisition of an MS3 spectrum. By ETD no neutral loss of phosphate was observed, but rather each phosphorylated residue possessed a unique mass, such as 243 Da for phosphotyrosine, allowing facile data interpretation.
Overall, an increased and quantifiable ability to detect and identify sites of phosphorylation was observed for ETD relative to MSA. However, certain peptides were best identified by ETD (smaller m/z and more highly charged) and others were best identified by MSA (higher m/z and doubly charged). In summary, the fullest characterization of a recombinant kinase could be achieved by the use of multiple dissociation techniques.
Conventional collision-induced dissociation (CID) has drawbacks in terms of a limited applicable molecular weight and the dependence of the fragmentation efficiency on the individual bond strength and amino acid sequence. Electron transfer dissociation (ETD) and its related proton transfer reaction (PTR) are alternative ways of fragmenting peptides and proteins, mostly applied in ion trap systems.
ETD became the preferred method for the analysis of common post-translational modifications (PTM) in proteins. Reversible phosphorylation is known to be one of the most important functions in eukaryotic cells, but its detection and characterization is often difficult. In most cases, the conventional CID results in the neutral loss of phosphoric acid, resulting in missing information about its binding site and sometimes in low further fragmentation of the peptide chain itself. ETD keeps the labile PTM bonds intact, in contrast, by generating a prompt dissociation at the amino acid backbone, and therefore allows the facile localization of the PTM. For fragmentation via ETD, an excess of radical anions—generated in a negative chemical ionization source—is added to multiply charged peptide cations in the ion trap.
PTR is a most useful addition for the fragmentation of larger peptides or even small proteins in the top-down or mid-down approach. Intact proteins can be identified and/or sequenced without any prior enzymatic digestion. While ETD is still used for the dissociation itself, the PTR anions reduce the charge states of the highly charged fragments into “ion trap readable” numbers.
Presented here will be PTM elucidation of peptides and intact proteins in mixtures. N-terminal sequencing of intact proteins allows direct identification by database search. An interesting model for the usefulness of these techniques is, e.g., histones, whose biological function strongly depends on the attached modification.
The full characterization of glycoproteins is a challenge for modern analytical techniques. First, glycosylated structures often show a large variation in their glycosylation pattern, complicating their structural characterization. Those structures are also quite difficult to separate by conventional LC methods. Finally, glycopeptides are often present only at lower abundance in comparison to unmodified proteins, due to distribution over various molecular species.
Finally, the characterization of glycosylated peptides with MS/MS using collision-induced dissociation (CID) is generally impaired by the preferential fragmentation of the glycosidic bonds, while the peptide bonds are more stable and stay intact.
Electron transfer dissociation (ETD) in ion traps enables selective fragmentation of the peptide backbone while leaving the carbohydrate structure intact. Thus, a combination of ETD and CID provides completely complementary information, usable for a full characterization.
Applying high-resolution CID in a QTOF has the additional advantage that even larger peptides and their fragments can be fully isotopically resolved. Also, the high mass accuracy helps to interprete the usually quite complex sugar moiety cleavage spectra.
Presented here will be the combination of high resolution CID and trap ETD on intact serum amyloid P component (SAP) proteins. SAP is a 25-kDa subunit pentameric glycoprotein identified as part of the fibrillar deposits in amyloidosis, e.g., in Alzheimer’s disease. The protein belongs to the pentraxin family of calcium-dependent carbohydrate-binding lectin. The physiological function of SAP is unknown, but its association with all types of amyloid may suggest chaperone functions, and we thus are studying ligand binding and structure of SAP using high-resolution techniques. In a first run, LC-autoMS/MS-CID is used to screen for glycopeptides via typical fragment ions like those at m/z 204, 366, 657, etc. In a second run, ETD is performed on the listed precursor ions for the elucidation of the glycosylation site and protein identification.
Performing post-translation modification (PTM) analysis by tandem mass spectrometry is still not a routine task, as there are an enormous variety of possible PTMs, their exact position in the sequence needs to be accurately determined, and the absence of a universal widely accepted probability-based scoring threshold makes it a time-consuming expert task to carefully validate results. A new generation of second-round search engines like MODIRO are dedicated for PTM analysis only, and limit the search to the already identified proteins, to overcome these bottlenecks.
Here, we compare widespread first-round search engines including MASCOT, XTandem!, and Sequest with the second-round algorithms MODRIO and MASCOT for the ability to detect phosphorylation on highly phosphorylated proteins in cancer pathways. Data-dependent LC/MS/MS data were acquired using a Thermo Scientific LTQ linear ion trap from a gel-digested protein.
We found that the ability to find phosphorylation is not primary related to the type of search algorithm (first- or second-round approach), but rather to the algorithm itself, and we found significant differences in the relative ability to detect phosphorylation sites from the same dataset.
Additionally, we could identify phosphorylation sites not annotated in SwissProt or previously identified according to the Phosphosite database (Cell Signaling Technologies), and validated these sites by score cut-offs, manual inspection of fragment ions, as well as cross-validation by several other algorithms.
Protein identification and the characterization of post-translational modifications (PTMs) by mass spectrometry is often limited by poor sequence coverage and labile post-translational modifications. Sequence coverage can be improved through the parallel use of multiple pro-teases to generate overlapping, complementary peptides, and electron transfer dissociation (ETD) to fragment larger peptides without disrupting labile PTMs. In order to evaluate the relative benefits of multiple proteases and multiple MS/MS fragmentation methods, we combined these approaches and assessed the sequence coverage and PTM mapping of albumin, horseradish peroxidase, and Erk. Our analysis workflow included a fluorescent protein digestion indicator to monitor the digestion progress, and MultiConsensus reports in Bioworks 3.4 to analyze and combine the data from multiple proteases and fragmentation methods. The combination of multiple digestion and fragmentation approaches improved the sequence coverage and PTM identification when compared to analyzing the same samples using trypsin and CID. This analysis also helped us identify the proteases and fragmentation methods likely to be most complementary for the analysis of complex protein samples.
Glycosylation of the serum immunoglobulin G (IgG) constant region is known to change with age, pregnancy, as well as various diseases. We here describe a method to monitor Fc-part N-glycosylation profiles for IgG1, IgG2, IgG3, and IgG4 within large study cohorts. IgG is captured from 2 μL of serum using protein A (binds IgG1, IgG2, and IgG4) and subsequently protein G (binds IgG3 as well) at the 96-well plate level, which is suitable for automation. Isolated IgGs were digested with trypsin, and obtained glycopeptides were analyzed by nano-LC-MS. Glycopeptides were characterized by collision-induced dissociation (CID) as well as electron-transfer dissociation (ETD) on a three-dimensional ion trap MS. These two fragmentation techniques were performed online in alternating mode. CID predominantly resulted in the fragmentation of glycosidic bonds, while ETD resulted in c-type and z-type cleavage of the peptide moiety. In combination, these techniques allowed the detailed characterization of the registered glycopeptides. The method is currently used to analyze IgG glycosylation at the subclass level within patient cohorts from various autoimmune diseases.
Phosphorylation is an important post-translational modification, for it affects signal transduction, malignant transformation, and other cell dynamics. However, research on phosphorylation doesn’t advance rapidly, for phosphoproteins are present in small amounts in the cell and are not detected with high sensitivity under mass spectrometry analysis. So enrichment technology of phosphopeptides is a very important issue. Titanium dioxide sorbent is already used by many researchers for enrichment of phosphopeptides and provides good results compare with IMAC technology. However, not all users get a gratifying result. So we have developed a new kit, which includes a titanium dioxide column and reagents. We have also optimized surface activity of the titanium dioxide sorbent used this kit. We will present comparison data of titanium dioxide and IMAC technology, the new kit, and current titanium dioxide sorbent.
Measuring the subtle physical behaviors of biomaterials at the solid:liquid interface is a key aspect in understanding and exploiting their performance, and can lead to more effective synthesis and fabrication of new biomaterials. Recently, dual polarization interferometry (DPI), an optical biophysical technique, has been used to characterize a number of biomolecular interactions relevant to a wide range of applications, from proteomics to tissue engineering. DPI provides structural information on both immobilized biomolecules and materials impinging on them at the solid substrate interface. Measurements of geometric molecular thickness and density enable researchers to probe structural changes to extremely high resolution (thickness sensitivity to 0.01 nm and surface concentrations of 0.1 pg/mm2) in real time. The responses of various biomaterials as a result of stimuli including protein and lipid interaction, metal-ion binding, antigen screening, and localized pH, temperature, and salt effects have been investigated.
Quartz crystal microbalance with dissipation (QCM-D) is a technique whereby the oscillation frequency of a coated quartz disc and the rate at which the oscillation energy decays are monitored to produce information about the mass and viscoelastic properties of an adsorbed material. Capable of operating in liquids, QCM-D provides a powerful approach to analyze the in situ structural and viscoelastic properties of biomolecules under near-physiological conditions. This discussion will focus on the application of QCM-D for nanoscale characterization of biological materials, including DNA, proteins, vesicles, and cells, as well as their interactions with other biological materials, polymers, or inorganic surfaces. This includes monitoring DNA hybridization and protein-protein, protein-substrate, vesicle-substrate, and cell-substrate interactions. We will also describe several “multimodal” QCM-D platforms that combine the technique with complementary analytical techniques to further derive information about complex biological systems.
Sample complexity is a key challenge facing proteomic analysis. New developments in column technology allow rapid, improved-resolution MS-based identification of intact proteins from complex samples.
Here, we separate complex mixtures, such as bacterial lysates, eukaryotic parasite, and transformed human cell lines, using online 2D LC on derivitized polystyrene-divinylbenzene pellicular ion-exchange resins and reversed-phase monolithic columns. Proteolytic digestion of fractions, followed by rapid LC-MS/MS, was used to complete the analysis. Alternatively, direct analysis of the second dimension eluents by top-down methodology, using Fourier transform ion cyclotron resonance mass spectroscopy (FTICR-MS), has allowed identification from intact Leishmania proteins and PTM mapping of histone H4.
Lysate separation was performed using SAX, followed by monolithic RP using rapid gradients. Second-dimension fractions were collected in 384-well microtiter plates and subjected to trypsin digestion.
Use of parallel 200-μm monoliths for tryptic peptide separations ensured maximum capacity, minimum sample loss, and high sample throughput, without sensitivity loss. For simple mixtures, RP separation times can be reduced to a few minutes, without significantly affecting data content.
Analysis of the digested fractions gave good coverage of the proteome, identifying proteins representing low (8 kDa) and high (500 kDa) molecular mass and extremes of predicted pI, as well as a number of membrane proteins. Resolution of intact protein separations was such that single protein species often occurred in one or two fractions for both the ion-exchange and reversed-phase separations, with fractions varying in complexity. Separation of modified proteins in the ion-exchange dimension demonstrated separation of isoforms.
Quantitation is of paramount importance to any proteomic technique, and LC separation of intact proteins provides unparallelled flexibility for differential analysis of complex samples. UV-absorbance maps were generated and used for differential analysis of samples. Isotopic labeling has been employed for more in-depth analysis of quantitative differences between samples, as well as label-free techniques for protein quantitation by LC/FTICR-MS.
Liquid chromatography plays a major role in purification, characterization, and QC analysis of therapeutic proteins such as monoclonal antibodies (mAbs). Protein purification often requires a combination of chromatographic techniques to yield the desired product purity. Commonly applied chromatographic techniques include affinity separations on protein A or G columns followed by size exclusion chromatography. Further purification is achieved by ion-exchange chromatography. Protein analysis and characterization is essential to steer the production process and assure product consistency.
Here we describe the application of a biocompatible LC system for purification and analysis of biopharmaceutical proteins. The column compartment with two dual-position switching valves in combination with a dual gradient pump allows for multidimensional separation schemes. The flexibility and robustness of the system is demonstrated by the following examples: two-dimensional peptide mapping of mAbs, stability testing of intact proteins, and ion-exchange purification and subsequent RP-HPLC-MS characterization of protein variants.
Recombinant monoclonal antibodies (MAbs) are becoming increasingly important as effective therapeutic proteins. Although the amino acid sequence and disulphide bond patterns are known, the MAb can undergo changes in enzymatic and non-enzymatic reactions during expression, production, purification, and storage. Protein variants can result from C-terminal lysine truncation or post-translational modifications such as glycosylation, deamidation, and oxidation. In addition, aggregation of proteins is a critical problem in therapeutics. The pharmaceutical activity and stability of the MAb is directly influenced by these reactions, and extensive characterization of protein variants is thus a prerequisite for successful development of pharmaceutical protein products.
Liquid chromatography is widely used in the quality control of purified, formulated therapeutic proteins such as MAbs. Ion-exchange chromatography is ideally suited for protein variant separations, as protein modification often leads to a change in charge distribution on the surface of the protein.
In this contribution we discuss the use of particulate ion exchange columns such as ProPac WCX-10 and monolithic ProSwift WCX-1S columns technology for the separation of charge variants of MAbs. We also present the advantages of monolithic columns for specific applications. The protein separations are performed on a new biocompatible LC system with a flow path composed of titanium and PEEK.
Identification of proteins in a vastly complex biological mixture presents a formidable challenge for analysis that no single separation protocol can satisfy. As a result, there is a need for sample-specific separation methods to achieve high-resolution and high-recovery results for downstream analysis of these samples. Currently, a standard technique for resolving protein mixtures, prior to LC/MS/MS analysis, is two-dimensional polyacrylamide gel electrophoresis (2D PAGE). However, this technique offers poor recoveries and relatively limited resolution and sensitivity, with only mid- to high-abundant proteins detectable. We investigate an approach that couples an OFFGEL pI fractionation technique with an orthogonal superficially macroporous reversed-phase column separation. This approach offers a 2D PAGE–like separation but has the advantage of keeping the protein sample in liquid, avoiding the negative aspects of polyacrylamide gel workflows. In this study, we present an enhanced protein identification technique facilitated by both an OFFGEL method for separations of complex protein biological samples based on pI and a novel reversed-phase strategy for separating intact proteins; together, they comprise an automatable workflow for increasing protein identifications.
The initial stage of protein fractionation leverages accepted pI technology in the form of IPG strips. The same mechanism is employed to effect the separation, the difference being that the protein sample is suspended and collected in liquid. This greatly facilitates downstream sample handling and does not limit possible workflows. The liquid fractions are collected and, with minimal sample preparation, injected onto a capillary superficially macroporous column. With use of the macroporous column and optimized conditions, we have demonstrated enhanced peak resolution, achieved high recoveries >98%, obtained high run-to-run reproducibility, and permitted higher column load tolerances than traditional porous column materials. The capillary HPLC is interfaced to a QTOF mass spectrometer via an orthogonal ES interface for protein identifications.
A major bottleneck in downstream proteomic analyses by 2D-GE and LC/MS is the initial complexity of the samples. With dynamic ranges of the analytes as broad as 1016, it is impossible to detect low-abundant proteins without initial complexity reduction of the sample. Robust, reproducible, and quantitative tools for complexity reduction enable successful ID and abundance analyses. Understanding regulation of protein activity by phosphorylation is a key to understanding many cellular pathways, yet a relatively small percentage of proteins are phosphorylated in any given cell. Enriching for phosphorylated proteins before 2D-GE or LC/MS analyses can increase the sensitivity of the assay and allow for the detection of lower-abundance proteins. We present here an IMAC resin that binds proteins with phosphorylated serine, threonine, and tyrosine residues. One mL of resin allows us to enrich up to 4 mg of total phosphorylated proteins from cell lines or tissue samples of interest in less than 1 h. Multiple cell lines were extracted and subjected to chromatography on the resin and various amounts of phosphorylated proteins were recovered quantitatively, as verified by western analyses with some target phophospecific antibodies. The data confirm that all three major groups of phosphorylated side chains are enriched quantitatively and reproducibly.
Detection of proteins using chemiluminescence-based western blotting is a well-known technique, where the signal is traditionally captured by exposing the membrane to film. Although it is a sensitive technique, the dynamic range is limited, thereby reducing the accuracy for quantification. The ImageQuant imagers are a new family of CCD imagers designed to support quantitative analysis of western blots, matching the sensitivity of film but improving the dynamic range.
We here demonstrate the benefits of using the ImageQuant 350 imager for capturing a digital signal of your western blot. ImageQuant 350 offers a broad dynamic range, 16-bit cooled CCD, enabling detection of very low and very high signals in the same image, making it an optimal choice for detection using ECL reagents. In a comparison with film using ECL Plus, ImageQuant 350 achieved sensitivity similar to that of film but with a significant improvement in dynamic range. The signal response for ImageQuant 350 was linear over the complete range of protein concentration. The combination of ImageQuant 350 imager with ImageQuant TL is a powerful tool for accurate relative quantification of signals.
In addition to chemiluminescent applications, ImageQuant 350 is useful for imaging of single channel fluorescence- and colorimetric 1D gel applications. ImageQuant imagers are compatible with various protein and DNA gel stains, which further demonstrates the broad application possibilities of these imagers.
Phosphorylation is a common reversible post-translational modification (PTM) in eukaryotes that is involved in regulation of essentially all biological processes—sub-cellular localization, enzymatic activity, protein complex formation, and many other functions.
Immobilized metal-ion affinity chromatography (IMAC) is a common technique used for purification/enrichment of phosphopeptides. Phos SpinTrap Fe is a new kit based on Fe Sepharose 6 Fast Flow immobilized with Fe3+. The specificity towards a model peptide from trypsinized bovine beta-casein spiked into a background of albumin tryptic fragments was tested. The enriched model phosphopeptide was analyzed with mass-spectrometric methods. The results were verified by repeating the experiment 10 times. The reproducibility was high and the signal strength from the phosphopeptide was increased from very low in the starting material to one of the strongest in the eluate. Finally, yeast extract was used as a complex sample and the trypsinized sample was run through Phos SpinTrap Fe. Several phosphopeptides were detected in the enriched fractions. Conclusions: Phos SpinTrap Fe is a specific kit for phosphopeptides that gives reproducible results.
The Affinity Reagents Group at the Pacific Northwest National Laboratory is focused on developing high-quality antibodies and protein-based affinity reagents for applications in biodetection, including biosensors and protein microarrays. We have built a strong foundation in the development of antibodies using yeast surface display, and have leveraged this background to create a platform for the rapid production of affinity reagents from both immunized and naïve sources. This antibody discovery platform, combined with our protein evolution and production capabilities, can rapidly produce quality, high-affinity detection reagents for use in human health, proteomic, diagnostic, and biodetection applications.
Edman sequencing has been the established method for determining the N-terminal sequence for proteins and peptides. However, this half-century-old technology has limitations with respect to time and cost effectiveness. Newer, alternate methodologies, such as mass spectrometry, have the potential to resolve questions traditionally answered by Edman sequencing. While the correct processing of a purified protein remains a solution best obtained from Edman degradation, we propose microwave-assisted acid hydrolysis (MAAH) followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis as an alternative high-throughput first-pass method for obtaining N- and C-terminal sequence information.
Microwave-assisted proteomics techniques have proven invaluable in recent years to accelerate many routine biochemical and enzymatic processes. MAAH has been shown to fragment the protein backbone, and for short incubations, renders predominantly N- and C-terminal peptides, which are analyzed by MALDI-MS. The mass spectrum is analyzed by an innovative, in-house bioinformatic platform that pairs the monoisotopic peak information to theoretical sequence fragments generated from the termini. In this manner, in minutes, one can determine whether a pure protein is properly processed.
MAAH followed by MALDI-MS and bioinformatics processing serves as a high-throughput screening technique, decreasing total analysis time and befitting a core sequencing laboratory.
The UC Davis Proteomics Facility offers protein analysis via amino acid analysis, Edman sequencing, and protein tandem mass spectrometry techniques, including protein identification, post-translational modification discovery, label-free quantitation, and protein cross-linking analysis. The facility utilizes state-of-the-art instrumentation, including a Thermo LTQ-FT Ultra. Our group utilizes open-source software (including X!Tandem and XCMS) and uses techniques and protocols that are made publically available. Using open-source software allows us to disseminate results to our users and allows users to analyze their own data without a significant monetary investment. We also offer data analysis and sample preparation classes to UC Davis and the community at large, including a week-long hands-on summer short course. Our group operates as an open core facility within the Genome Center on the UC Davis campus.
The mission of Proteomics Resources is to establish a fully integrated core facility for proteomics analyses at Roswell Park Cancer Institute (RPCI). There are a variety of electrophoresis-based, HPLC-based, and mass spectrometry–based capabilities for proteomics applications, and these can be variously configured depending on the needs of the particular project. Being a part of the shared research resources and services available at RPCI, Proteomics Resources interacts with other proteomics facilities that are currently being developed at the University at Buffalo. Together, these resources provide essential support for proteomics projects in Buffalo. The resources also provide training and education programs to the faculty, staff, and students of RPCI and SUNY at Buffalo. http://www.roswellpark.org/proteomics
The University of Vermont/Vermont Genetics Network (UVM/VGN) Proteomics Facility is an interdisciplinary core facility in collaboration with the Departments of Biology and Chemistry, and the College of Medicine, at the University of Vermont. The facility is funded by VGN through NIH (grant P20 RR16462).
The facility provides services in protein identification, characterization of post-translational modifications (e.g., methylation, phosphorylation), and quantitation of proteins and their modifications. The goal of the facility is to offer a reliable, efficient, and effective service for proteomics research and the education needs of the UVM and other institutions.
The facility has four mass spectrometers, including one matrix-assisted laser desorption-time of flight mass spectrometer (MALDI-TOF-MS), one LCQ Deca XP ion trap mass spectrometer plus liquid chromatograph (LC-MS), and two LTQ linear quadrupole ion trap mass spectrometers (nano-LC-MS).
The facility is led by Professor Dwight Matthews, who directs mass spectrometry instrumentation in several locations at UVM. Our facility manager, Dr. Deng, interacts with investigators and provides scientific support to the proteomics projects. Dr. Mark Jennings develops proteomics methods and provides technical support for users.
The facility has run thousands of samples since last year, which included profiling expressed proteins in tissues, fluids, and cells, mapping sites of protein phosphorylation and other post-translational modifications, and detecting target proteins in biomedical samples.
The Protein Discovery Initiative goal is to develop a census of the migration proteome by identifying interactions and post-translational modifications, i.e., phosphorylation, of novel molecules involved in cell migration. Over the past few years, identifications of these phosphorylation sites have been done by a variety of methodologies, such as mutation of phosphorylation sites, phospho-antibodies, electrophoretic mobility shifts, immobilized metal ion affinity chromatography (IMAC), titanium dioxide (TiO2) affinity chromatography, and LC-MS/MS. Over the past year, we have developed a standard approach for phosphopeptide enrichment using direct LC-MS/MS and TiO2 affinity chromatography coupled with LC-MS/MS.
Reversed-phase chromatography in C18 column was used to analyze a tryptic digest of Wiskott-Aldrich Syndrome Protein (WASP). The phosphopeptide enrichment method modified from Cantin et al.1 with or without the addition of 2,5-dihydroxybenzoic acid (DHB) was used to isolate and identify phosphorylation sites. Protein coverage and spectra counting of these three runs were used to compare the efficiency of the different methodologies.
Finally, we compared phosphorylation sites identified in the Protein Discovery Initiative with results found in databases (PhosphoSITE, http://www.phosphosite.org, and Phospho.ELM, http://phospho.elm.eu.org). Thus far, using our established methodologies, we have found a number of phosphorylation sites in the proteins studied that were not reported in the other two databases.
The Vanderbilt Mass Spectrometry Research Center (MSRC) provides an integrated bioanalytical service facility to the university research community. It offers various state-of-the-art proteomics, tissue profiling/imaging and bioanalytical MS shared facilities. These cores are managed by a professional staff of seven faculty members and eleven research assistants, bioinformatics specialists and an instrument engineer. The Proteomics Laboratory supports multiple technology platforms, including multidimensional HPLC peptide separations and 2D gel separations of intact proteins, followed by ESI-linear ion trap (including Orbi-trap) and MALDI-TOF/TOF mass spectrometry and supporting bioinformatics algorithms for protein identification and characterization of protein post-translational modification. We routinely utilize single- and multi-dimensional LC/MS/MS for protein cataloguing and differential-expression studies (using spectral counting), and difference gel electrophoresis (DIGE) for large-scale, multiple-variable expression studies on simple and complex proteomes. The tissue imaging core provides tissue sectioning, staining, and MS directly from tissue sections. This can be accomplished at high resolution (imaging) where the entire tissue section is interrogated and ion density maps of proteins are reconstructed, or in a high-throughput histology-directed profiling approach where specific areas are targeted. Both of these cores work closely with users at all stages of experiments including detailed post hoc informatics consultations, but generally operate as limited-access facilities where users prepare samples and core technical staff performs the analyses. The bioanalytical MS core provides instrumentation to perform a wide variety of analyses (e.g., identification and structural analysis of biological molecules, qualitative and quantitative assays of drugs and metabolites). The MS core operates in an open access environment where users (usually students of fellows) are encouraged to run their own samples with the advice and assistance of Core personnel. The MSRC also offers a variety of educational instrument operation and training classes throughout the year to facilitate optimal core usage.
The Charles W Gehrke Proteomics Center at the University of Missouri-Columbia is a full-service core facility providing two-dimensional gel electrophoresis and mass spectrometry services. We accept any nonhazardous and nonradioactive samples.
For 2D gel analysis we accept whole-tissue samples, partially purified samples (e.g., immunoprecipitations, column fractions, etc.), and purified acetone-precipitated protein samples. We routinely employ the difference gel electrophoresis technique for quantitative protein expression profiling. Expression analysis can be conducted using DeCyder and Phoretix software. Additionally, we offer total, phospho-specific, glyco-specific, and integral-membrane-specific post-staining of 1D and 2D gels. We also have electrophoretic transfer apparatus for immobilizing proteins on nitro-cellulose of PVDF membranes for gels up to 16 cm2.
For mass spectrometry services, we accept 1D bands, 2D spots, purified proteins in solution, and can also prepare protein from tissue for quantitative profiling. Our mass spectrometry services include intact mass analysis on proteins up to 100 kDa using our Applied Biosystems Voyager DE-PRO, trypsin (and other protease) digestions, MS and MS/MS analyses on our Applied Biosystems 4700 TOF-TOF and Applied Biosystems QSTAR pulsar QqTOF. Protein identifications are facilitated using in-house copies of MASCOT and ProteinProspector. We offer iTRAQ expression analyses using either the TOF-TOF or QSTAR, both of which are integrated with nano/capillary-flow HPLC systems from LC Packings/Dionex.
Our mission is to provide cost-effective, timely, and validated data. Much of our staff time is spent educating our clients in project design and data interpretation. We provide detailed data for clients to allow them to meet the emerging standards for publication of proteomics data. Fee schedules and other useful information are posted on our Website: www.proteomics.missouri.edu.
The Proteomics and Mass Spectrometry Facility of the Cornell University Biotechnology Center provides an array of shared research resources and services to the university community and to outside investigators on a fee-for-service basis. The mission of the facility is to provide faculty, students and researchers with cutting-edge technologies in mass spectrometry and proteomics, high-quality services, and innovative solutions that will significantly contribute to the campuswide life sciences research, training, and education programs.
The facility is well equipped with advanced mass spectrometers for both proteomics and metabolomics analyses. We have four mass spectrometers, including a MALDI-TOF-TOF (4700) and a triple-quadrupole linear ion trap (4000 Q Trap), both from Applied Biosystems. Also, we have three HPLC systems, including a 2D nano-HPLC system from Dionex with a built-in automated spotter robot (Probot). We also have two 2D gel systems and a powerful laser scanner, Typhoon 9400 from GE Healthcare, as well as two robotic units for high-throughput gel picking and liquid handling (ProPic and ProPrep).
We offer services including 2D gel and 2D LC separation for macromolecules, robotic or manual sample preparation for proteomics-associated samples, protein identification, quantitative proteomics by 2D DIGE analysis or shotgun-based iTRAQ, and protein characterization on post-translational modifications through both nontargeted discovery and targeted discovery approaches. We also offer MS data interpretation through several bioinformatics tools as well as small-molecule identification and quantitation.
Peptide identification from high-throughput tandem mass spectrometric data is increasing in reliability and reproducibility, but even with validated peptides, the assembly into a set of proteins can be ambiguous. Using the original search results to identify proteins typically results in overcounting of the proteins present. There are several protein identification programs that attempt to determine the minimum number of most likely proteins, but their output uses an awkward method of handling peptides common to different proteins by replicating them for each protein, resulting in related proteins distributed throughout the profile. In addition, comparison of samples and handling of large datasets can be difficult. IsoformResolver (IR) is protein identification software developed in our core facility that was designed to compare and make sense of very large datasets. Using validated peptides from various search program inputs, IR reconstructs the protein identifications, independent of those generated by the search program, using a novel peptide-centric database. Protein groups are generated by logically connecting proteins with peptides in common, resulting in groups of proteins that are primarily splice variants and paralogs of each other. This approach removes the need to replicate peptides and organizes the protein profile in a manner that removes ambiguities and redundancies. IR can generate profiles comparing any number of samples, and includes protein annotations (genes, GO categories, and identifiers from numerous databanks), removal of unlikely peptide isoforms, and automatic reporting of label-free quantification using spectral counts in a summary output convenient for further analysis. Results will be shown for protein profiles using various datasets, ranging from protein standards to fractionated whole-cell lysates, and IR output will be compared to output from several popular free and for fee protein identification programs.
Low-molecular-weight proteins in blood plasma and serum, such as chemokines, and antimicrobial peptides play an important role in controlling immune responses, angiogenesis, and many other physiological processes. The concentration of these molecules in plasma is usually determined using antibody-based methods such as ELISA, but the precise molecular form (e.g., actual N-terminus) of these polypeptides, which determines their activity, cannot not be easily determined. Proteomics analysis based on mass spectrometric identification of peptides after digestion with trypsin or other proteases, usually does not allow detection of chemokines in blood serum or plasma dominated by large abundant proteins such as albumin.
Previously, we have demonstrated the effectiveness of organic solvents such as acetonitrile and isopropanol in the presence of the ion-pairing agent trifluoroacetic acid for extraction of low-mass proteins from small volumes of serum. We wanted to investigate what chemokines and other regulatory polypeptides we can directly detect in human blood plasma and serum using organic solvent extraction in combination with reversed-phase liquid chromatography, N-terminal protein sequencing, MALDI-TOF, and ESI MS.
Using these techniques, we have detected and identified platelet factor 4, C5a, C4a, C-TAP, and SDF-1; therefore, we have demonstrated that the regulatory polypeptides could be directly isolated and biochemically characterized from small samples of blood plasma or serum.
Funded by NCI Contract No. N01-CO-12400.
Differential (fluorescence) gel electrophoresis (DIGE) is a powerful tool that utilizes 2D SDS-PAGE for locating proteins that change in expression due to treatment/disease state. By incorporating mass- and charge-matched fluorescent dyes, three samples can be compared in one 2D gel, thereby eliminating spot misalignment. Using the DeCyder software (GE Healthcare), protein spots are detected and quantitated on a pair (or group) of fluorescent images, leading to the determination of which protein spots change abundance. Selected proteins are then identified by robotic excision of the gel spot, tryptic digestion, and mass spectrometric analysis. One challenge with DIGE is efficient presentation of the gel image, spot location, and protein identification. Open-source software called Yale Protein Expression Database (YPED) was developed within our resource to help solve this challenge. YPED names protein spots from MALDI-TOF/TOF analysis (AB 4700/4800), and links gel images with the proteins identified from the mass spectrometric analysis. Users log on a Web interface using their unique logon ID and password. By choosing the experiment of interest, they can see the gel image with identified spots indicated. Clicking on the spot lists the protein spot number from DeCyder, the protein identified, the corresponding peptide sequences, Mascot scores, Cy dye ratios, etc. Data are exportable in Excel format. A link to the Panther Classification System aids the researcher in studying pathways and functions. The power and benefit of DIGE is most clearly evident in looking at sites of post-translational modifications (PTM). By utilizing a metal oxide enrichment step prior to DIGE analysis, we are able to locate phosphoproteins, as well as changes in the phosphoproteome. DIGE can also detect changes in protein glycosylation, particularly when comparing a sample before and after deglycosylation. Examples of both types of PTMs will be shown.
Mutations in the gene coding for the neuronal protein a-synuclein (aS) have been linked to familial Parkinson’s disease (PD). S129 phosphorylated aS has been shown to be highly enriched in Lewy bodies, a pathological hallmark of PD. Our lab has also shown that the C-terminus tail of aS can be phosphorylated on Y125. However, little is known of the normal function of aS phosphorylation. In this work, we use a mass spectrometric–based targeted functional proteomic approach to identify qualitative and relative quantitative differences in protein-protein interactions of the phosphorylated versus nonphophorylated C-terminus of a-synuclein.
Biotinylated peptides of the aS C-terminus tail have been synthesized as nonphosphorylated, S129 phosphorylated, Y125 phosphorylated, and a scrambled sequence as a control for nonspecific binding. Protein pull-down assays are performed on streptavidin-coated magnetic beads, where the bound peptide is incubated with mouse brain synaptic lysate. Eluted proteins are separated by SDS-PAGE. Each lane is cut into 40 slices, in-gel digested, and analyzed by LC-MS/MS followed by protein sequence library searches for peptide assignments. All pull-down assays are performed in triplicate with different pooled lysates. To facilitate parsimonious assignment, comparison of datasets, and relative quantification of peptide and putative protein assignments across these large datasets, both for a given gel and across gels, in-house software (MassSieve) was developed.
These data provide evidence that nonphosphorylated aS preferentially interacts with mitochondrial electron transport chain proteins, while phosphorylated aS interacts with cytoskeletal proteins. This suggests a change in function of aS upon phosphorylation.
We have previously demonstrated primary neuronal SILAC (stable isotope labeling by amino acids in cell culture) as a powerful tool for applying quantitative proteomic approaches to the study of neuronal molecular and cellular dynamics. Here, we have applied this technique to assess changes of the neuronal phosphotyrosine proteome in response to the neurotrophins BDNF and NT-4. These two proteins function as ligands for the receptor tyrosine kinase TrkB. Interestingly, they have been shown to have similar affinities for the receptor and demonstrate equal activation of the receptor as measured by receptor phosphorylation, but the two ligands induce differential downstream signaling and receptor trafficking outcomes. Phosphotyrosine immunoprecipitates from triple-condition primary neuronal SILAC experiments (NT-4 treated vs. BDNF treated vs. control) were fractionated by 1D SDS-PAGE, analyzed via LC-MS/MS, and a total of 700 proteins were identified via Mascot. SILAC ratios indicated that 31 proteins increased their abundance in phosphotyrosine immunoprecipitates under both stimulation conditions as compared to controls, pointing to shared signaling components. SILAC ratios also showed that 19 proteins were more abundant in immunoprecipitates from BDNF treated but not NT-4 treated neurons, and 66 in NT-4 treated but not BDNF treated neurons, suggesting a number of explanations for differential signaling through a single receptor. This includes differential engagement of other families of receptor tyrosine kinases, regulation of motor proteins and trafficking machinery, and cytoskeletal rearrangement.
In eukaryotic cells, post-translational modifications such as phosphorylation are involved in numerous signal transduction pathways that control cellular proliferation, differentiation, and apoptosis. The comprehensive characterization of phosphorylation sites within a single pathway remains a challenge to the proteomics researcher. Current enrichment procedures such as immobilized metal ion affinity chromatography (IMAC) show minor nonspecific binding and low recovery of lower-abundance phosphopeptides, in particular phosphotyrosine. In addition, each enrichment technique demonstrates a different specificity. Immunoprecipitation strategies have proven useful for enriching phosphotyrosine-containing peptides in the literature, and IMAC has been used on the eluate to further that enrichment. We propose a combination of enrichment methods to improve the recovery of phosphopeptides and generate a more complete phosphoproteomic picture that has been termed Y-MAC. During Y-MAC, the flow-through after phosphotyrosine immuno-enrichment is subjected to IMAC and subsequent TiO2 enrichment procedures.
We present the phosphoproteomic analysis of a stimulated cell lysate using Y-MAC. The lysate was first phospho-enriched by immunoprecipitation using a phosphotyrosine-specific antibody. The eluate and flow-through fractions were separated by SDS-PAGE, and in-gel digestions using trypsin were performed. Phosphopeptides in the flow-through fractions were enriched by IMAC and further with TiO2, then analyzed by LC-MS/MS. The eluate, which contains the phosphotyrosine-enriched proteins, was analyzed by LC-MS/MS after in-gel tryptic digestion. Y-MAC allows a more comprehensive phosphoproteomic evaluation than can be obtained by any of these techniques alone.
The genomics and “immunologics” of F344-to-Lewis and Lewis-to-WR.1L rat models of chronic kidney rejection have been extensively studied. Renal allograft rejection in Lewis-to-WF.1L appears to be based on mismatch in minor histocompatibility loci, leading to changes in functional and histological features. In this study we investigate differences in kidney protein expression in these rat strains. We postulate that differential expression influences cellular defense mechanisms following allo-recognition.
Kidneys of 8- to 10-wk-old male F344, Lewis, and WF.1L rats (n = 3/strain) were freeze-clamped and used for 2D gel proteomic analysis. Statistically significant changes in protein abundances were determined with Image-Master software. Spots of interest (F344: 3; Lewis: 7; WF.1L: 9) from three replicate gels were excised, pooled, digested, and identified by tandem mass spectrometry and database search (Agilent Ultra ion trap; SpectrumMill and Mascot; SwissProt database).
The following proteins appear to be differentially expressed between the three strains: Ado-hyc-ase was observed only in Lewis rat kidneys. GPx3 and IDH were overexpressed in WF.1L as compared to Lewis and F344. ALDH2 was up-regulated in F344 and WF.1L compared with Lewis (all with p < 0.05, one-way ANOVA, Holm-Sidak Test). The data are displayed in the table below as the mean ± SD of the normalized %Vol. Results were confirmed by Western blotting.
Our proteomic approach identified significantly different expression of four proteins, two of them (ALDH2, GPx3) directly involved in cellular defense mechanisms against oxidative stress, one (IDH) primarily affected by oxidative stress. Based on our results, we speculate that these two models have different intracellular defense mechanisms against oxidative stress once allo-recognition has occurred.
Snake envenomations cause multiple effects on hemostasis. Local and systemic bleeding are attributed to the effect of hemorrhagic metalloproteinases on degradation of endothelial matrix. Jararhagin is a 52-KDa P-III met-alloproteinase isolated from the Brazilian pit-viper Both-rops jararaca. Jararhagin is a well-studied protein, able to cause hemorrhage as a result of membrane degradation and inhibition of platelet adhesion. Jararhagin interacts directly with von Willebrand factor, fibrinogen, and platelet integrins. Jararhagin is also known to cause a strong pro-inflammatory response with recruitment of leukocytes.
This work aims to evaluate the ability of jararhagin to alter protein expression in muscle fibers, using laser capture microdissection and mass spectrometry.
Groups of mice were injected intramuscularly with either saline solution (100 μL control) or jararhagin (5 μg/100 μL). At 15 min after injection, animals were sacrificed and the injected muscle was dissected out. Sections of 8 μm were cut, place in Director slides, and embedded in paraffin. Tissue sections were stained with eosin for laser capture microdissection (LCM). LCM was done in a Leica microscope in the Advanced Microscopy Facility (University of Virginia, UVA). The protein content of cells was extract using Liquid Tissue MS Protein Prep Kit. Three micrograms of cellular protein was labeled using iTRAQ Reagent Multi-Plex kit in three groups (control, high hemorrhagic, and low hemorrhagic tissue). Mass spectrometry analysis was done with Q-Star equipment at the Mass Spectrometry Core (UVA).
Laser capture microdissection allowed the precise collection of muscle fibers. The intracellular proteins were analyzed by quantitative proteomics using iTRAQ methodology, and 36 proteins could be identified. This work demonstrates that the combined approach of LCM and iTRAQ can be used in the identification of early cell damage and differential expression of individual cell types.
Rice blast, a catastrophe in rice production, is caused by the ascomycetous fungus Magnaporthe oryzae. This fungal pathogen is highly tractable and serves as a seminal model for the study of plant disease. Inactivation of the conidial morphology-moderating gene MgCOM1 in the fungal pathogen M. oryzae resulted in pleiotropic phenotypical changes, such as altered conidial morphology, reduced conidiation, suppression of penetration peg and infection hypha formation, and attenuated pathogenicity. A proteomics approach was employed to decipher these phenotypical changes at protein level, because proteins are directly related to function (phenotype). Proteins extracted from conidia of M. oryzae field isolate P131 and mgcom1– mutant were resolved on two pH scales (linear mode)—3–10 and 4–7. Eleven proteins were differentially regulated in the pH range of 3–10, and 20 in the pH range of 4–7. Among 31 protein spots that were differentially expressed on both pH scales, 10 proteins showed up-regulation in their expression level and 11 were down-regulated. Three protein spots exhibited expression only in null mutant, while seven proteins were not expressed in MgCOM1 mutants. These protein spots were subjected to mass spectrometric analysis after in-gel digestion. All 31 proteins were identified by MALDI-TOF MS. Peptide mass fingerprints were utilized for protein identification by analyzing the sizes of tryptic fragments via the Mascot (http://www.matrixscience) search engine using the entire NCBI fungal protein database. After mass spectrometry analysis and database search, 13 proteins were identified with known functions and the remaining 18 (hypothetical proteins, conserved hypothetical proteins, and predicted proteins) with unknown function. Proteomics analysis reveals that these differentially expressed proteins are associated with melanin biosynthesis, structural framework, lipid metabolism, glycolysis, TCA cycle, amino acid metabolism, protein modification, energy production, nuclear trafficking, transcription, phenylacetate catabolism, protein-protein or protein-DNA interactions, methionine metabolism, pre-mRNA splicing, methylation, molybdopterin-biosynthesis, cell regulation, leucine metabolism, and DNA replication.
There are currently no diagnostic indications that are always reliable, always obtainable, and always conclusive for pancreatic cysts. To establish more effective biomarkers for pancreatic cyst diagnosis, we used proteomics to perform a pilot investigation of the protein content of a series of pancreatic cyst fluids, and related the findings to clinical parameters, to provide deeper understanding about the molecular profile within these cysts.
Cyst classification required less than 40 uL of each sample. Over 500 different proteins were identified and quantified among the pancreatic cyst fluids. Besides comprehensive coverage of pancreatic enzymes and plasma infiltrate proteins, potentially effective biomarkers were discovered. Validation of these findings may enable proteomic profiling to become a valuable tool for identifying pancreatic cancer in patients presenting the smallest and earliest cystic lesions.
An important component of kidney research is the discovery of novel proteins that control cellular and molecular events that contribute to normal kidney cell biology and disease. Identifying perturbation of normal cellular protein expression is critical for understanding these regulatory events. Methods that couple two-dimensional capillary liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS) analysis have become an integral tool in this type of discovery science. We are using 2D LC-MS/MS combined with a unique label-free quantitative strategy to define protein expression differences in renal tubular extracts from OVE26 type 1 diabetic and control mice. Traditionally, quantitative mass spectrometry methods have employed stable-isotope labeling. However, these approaches are not generally used because of several limitations, including labeling artifacts, costs, throughput, and accessibility. Thus, we have developed a method that determines protein abundance without labeling. Overall, we have identified nearly 14,000 different proteins from replicate 2D LC-MS/MS experiments. Thus far, we have identified 476 differentially expressed proteins. These include a number of previously characterized phenotypic changes related to TGF- signaling and tubulointerstitial fibrosis. Also, many other diabetes-induced proteins are involved in glucose metabolism, and down-regulated proteins are primarily mitochondrial or have plasma membrane transport functions. These biologically relevant findings indicate the utility of using this method and an animal model for discovery of novel regulatory events in diabetic kidney disease. In addition, this technique allows for selective analysis of different components of the nephron and can aid in defining cell-specific regulation. We believe that this approach offers a robust, high-throughput, unbiased methodology that can be applied to any model of acute or chronic renal disease.
A common strategy for quantitative proteomics is to use liquid chromatography coupled to mass spectrometry. Accuracy and consistency of mass spectrometry can be improved through the use of a stable isotope-labeled internal standard. A method to obtain such a standard for every peptide in a complex sample is to metabolically incorporate a stable isotope during growth and combine tissue from test samples and the isotopically labeled standard sample prior to protein extraction, separation, tryptic digestion, etc. Here, such a method is implemented on a thermo-electron Orbitrap mass spectrometer. The method is characterized in terms of its precision and accuracy across a range of absolute signal intensities and peptide ratios. From this characterization, the potential statistical power of the technique is determined.
The proteins of the wheat endosperm, in particular the gluten complex, are of critical importance in determining the special viscoelastic properties of wheat flour dough. Because of the surprising complexity of the endosperm proteome, particularly with regard to the glutenin and gliadin regions, the effects of individual proteins on product quality are inadequately understood.
The aim of this study was to identify proteins associated with high dough strength and extensibility, with the ultimate aim of providing breeders with markers for further crop improvement and potentially new wheat quality assays. A proteomic approach was employed to study differences in the protein expression profiles of the endosperm of two wheat cultivars: one with high dough strength and extensibility and the other with low dough strength and extensibility. To narrow down the association, the study was extended to analyze a subset of a double haploid population derived from a cross of these parent lines.
The analysis was performed using two-dimensional gel electrophoresis with quantitative image analysis, over two pH ranges: pH 4–7 and pH 6–11. Nano-LC-ESI-MS/MS was used to identify protein spots unique or differentially expressed threefold or greater. The small number of differences identified in the acidic protein region (pH 4–7 gels) included unique alpha amylase inhibitor isoforms. The alkaline region (pH 6–11 gels) contained most of the differentially expressed proteins. As expected, the parents showed differences in the high-molecular-weight glutenin region of the gel. The low-molecular-weight glutenin region revealed two areas containing multiple spots unique to the high dough strength cultivar. Differences in spot profile were also found in two gamma-gliadin gel regions. The analysis of the progeny has revealed protein profile differences that can be related to phenotype.
Ripening in climacteric fruits, such as tomato, is regulated by multiple interconnecting pathways, some of which are regulated by the hormone ethylene, while others are under developmental control. The development of ripening-impaired tomato mutants has provided a set of valuable tools for dissecting this complex process. Three such mutants are ripening inhibitor (rin), never ripe (Nr), and non-ripening (nor). They exhibit severe phenotypes, since many of the pathways associated with normal ripening are disrupted. Here, we report a quantitative comparison of protein expression between wild-type tomato and these three near-isogenic mutants, to study the processes underlying fruit ripening. Total soluble protein extracts of tomato fruit pericarp from wild type and each of the mutants, taken at the same stage of the ripening process, were digested with trypsin, and each was labeled with a different iTRAQ mass tag. The samples were then mixed and fractionated by strong cation exchange chromatography. The fractions collected were then analyzed by LC-MS/MS using a Waters nano-Acquity UPLC coupled with a Synapt HDMS system. Proteins were identified and their expression quantified by using the mass spectral data to search a database of tomato proteins (SGN) with both Mascot (v. 2.2) and PLGS (v. 2.3). Over 700 proteins have been identified and functionally characterized. The biological implications concerning ripening-associated developmental pathways are discussed.
Changes in protein methylation, citrullination, and phosphorylation during experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis, were evaluated using isobaric tags for relative and absolute quantification analysis of peptides produced from normal and diseased rat lumbar spinal cords. We observed alterations in the post-translational modification of key proteins regulating signal transduction and axonal integrity. Dephosphorylation of discrete serine residues within the neurofilament-heavy subunit C-terminus was observed. We report for the first time elevated citrullination of Arg27 in glial fibrillary acidic protein, which may contribute to the pathophysiology of astrocytes.
Pancreas cancer often goes undetected until late-stage disease. Unlike prostate and ovarian cancer, no markers are available to detect pancreas cancer in earlier stages of disease. Plasma is a convenient source to detect potential biomarkers for cancer because individuals provide peripheral blood for other routine clinically indicated tests. We have identified a panel of peptides in 23 pancreas cancer patient plasma samples that are not present in plasma from 35 normal donors, using nanospray ion trap LC-MS/MS methods by searching custom peptide databases. High titer antibodies to several peptides were present in patient plasma samples, indicating that these peptides may be immunogenic as well as biomarkers. Next, we will analyze LC-MS/MS spectra from plasma of patients with pancreatitis, with the goal of differentiating pancreas cancer from other clinical conditions involving the pancreas.
One of the major hurdles in treating cancer patients is the inability to determine a patient’s response during the earliest stages of treatment. With death of tumor cells being the goal of cancer therapies, a non-invasive tool to measure the amount of tumor cell death during therapy would be invaluable.
Using the human tumor-SCID mouse xenograft model, we examined the changes in serum proteins that correlated with (1) the presence of a colon adenocarcinoma and (2) the response of that tumor to Apo2L/TRAIL (tumor necrosis factor–related apoptosis-inducing ligand). Sera were collected from a cohort of 12 mice prior to and 2 wks after the establishment of a subcutaneous Colo205 tumor. Samples were then pooled, depleted of albumin, and analyzed by differential in-gel electrophoresis (DIGE). Proteins with statistically significant differences (± twofold) were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and/or by liquid chromatography–electrospray ionization time-of-flight mass spectrometry. Tumor-bearing mice were then treated with a single dose of Apo2L/TRAIL and sera was collected from these mice 8 h later. DIGE analyses coupled with protein identification were performed as described above to compare sera from mice with tumors before and after Apo2L/TRAIL treatment.
Several acute phase proteins were found to be elevated in the sera from untreated mice with tumors as compared with controls, suggesting the generation of a host immune response. Interestingly, the animals treated with Apo2L/TRAIL showed no significant changes in the level of the acute phase proteins; however, a >threefold decrease in fibrinogen β-chain was observed. Future work will focus on understanding the significance of these findings, as monitoring these and other serum protein changes may be a useful tool for measuring the response of a tumor to a given therapy.
In 2D gel mapping, most protein spots consist of multiple proteins, posing a significant challenge for the proper interpretation of gel-based comparative experiments. Previously, we introduced an approach integrating 2D difference gel electrophoresis (DIGE) and LC-MS/MS analysis with the exponentially modified protein abundance index to correctly distribute a spot’s volume to each of its protein constituents. However, the experimental variation associated with the procedure was not fully established. Here, we evaluate the reliability of this method by analyzing replicate sets of samples.
Protein was extracted from an aluminum-tolerant cultivar of maize under Al-free and treated conditions and separated by 2D DIGE. After image analysis, spots of interest were excised, digested, and analyzed by nLC-MS/MS. Protein identifications were made, and associated emPAI values were generated for each spot by Mascot 2.2 through searching the maize database.
Twenty-seven matched spot pairs from the control and treated states were analyzed in duplicate. The results show that all contain multiple proteins, averaging 10 proteins/spot. The volume of each spot was distributed among its constituent proteins on the basis of their mol fraction, as determined by the emPAI. The fractional spot volume for individual proteins was compared between control and treated samples. Many instances were found in which down-regulated proteins were discovered in up-regulated spots and vice versa, demonstrating the importance of dissecting and distributing the change in spot volume. Approximately 90% of the protein identifications obtained from replicate spots matched, and their mol fractions were found to be nearly identical. Unmatched proteins and instances where the mol fractions of replicates deviated greatly were limited to the lowest-abundance proteins and do not impact the overall results significantly, suggesting the approach is reliable.
Carotenoids are a diverse group of pigments widely distributed in nature. They fulfill many essential functions in plants and play important roles in human nutrition and health. Much is known concerning the genes encoding the enzymes associated with carotene biosynthesis. However, little is known about the control mechanisms involved in carotenoid accumulation. It has been hypothesized that one such mechanism involves the formation of a “carotenoid sink” through the conversion of leucoplasts to chromoplasts, which function to sequester and stably store carotenoids. To identify the proteins involved in this conversion, we have compared the protein expression in leucoplasts isolated from white-type cauliflower to chromoplasts isolated from orange cauliflower by two-dimensional difference gel electrophoresis. Leucoplast and chromoplast protein samples were labeled with CyDye fluors. Two biological replicates were included for both leucoplasts and chromoplasts. Three technical replicates were carried out for each biological replicate. A dye swap was included in the experimental design to normalize for differences in the reactivity of the dyes. The gels were imaged with a Typhoon 9400 Variable Mode Imager, and the images were analyzed using Progenesis SameSpots. More than 150 protein spots were identified whose level of expression changed significantly between leucoplasts and chromoplasts, as judged by an ANOVA p value of <10-3 and a fold change of >1.6. These spots were excised from the gel, digested with trypsin, and analyzed by mass spectrometry using a model 4700 tandem time-of-flight mass spectrometer.
Our overall goal is to develop platforms that could be used for pre-symptomatic diagnosis. Key to this goal is inventing a new way to make antibodies. This process starts with screening proteins on printed microarrays of 10,000 random-sequence 20-amino-acid peptides. The kinetic properties of the individual peptides were studied by surface plasmon resonance. Pairs of peptides were selected by a process called SIPPA and incorporated into many synthetic scaffolds (multiple antigenic peptides, polyproline and proline-glycine-proline-based peptide scaffolds). Bivalent di-epitopic multiple antigenic peptides were synthesized via divergent peptide synthesis using orthogonal protecting groups on branched lysine. Varying lengths of polyproline and proline-glycine-proline-based peptide scaffolds were assembled via Cu (I)-catalyzed Huisgen reaction. These scaffolds were shown to have an order of magnitude increase in affinity compared to the individual peptides and Kds comparable to those of monoclonal antibodies.
The large-scale isolation and analysis of glycoproteins by lectin affinity chromatography coupled with mass spectrometry has become a powerful tool to monitor changes in the “glycoproteome” of mammalian cells. Thus far, however, this approach has not been used extensively for the analysis of plant glycoproteins. As with all eukaryotes, N-glycosylation in plant cells is a common post-translational modification for proteins traveling through the secretory pathway. Many such proteins are destined for the cell wall, or apoplast, where they play important roles in processes such as modifying cell-wall structure, sugar metabolism, signaling, and defense against microbes. We describe a strategy to isolate and identify secreted plant proteins based on lectin affinity chromatography and 2D LC-MALDI MS/MS analysis, and demonstrate how this approach can be used to study fruit ripening, a plant developmental process that is fundamentally linked with major qualitative and quantitative changes in the secretome. Total soluble protein extracts of tomato (Solanum lycopersicum) fruit pericarp were applied to a Concanavalin A Sepharose column. After proteolysis of the selected proteins, the peptide mixtures were separated using strong cation-exchange (SCX) chromatography. The SCX fractions were then separated with a nanoscale reversed-phase liquid chromatograph coupled to a MALDI plate spotting robot. These spots were analyzed offline by MALDI MS/MS. Proteins were identified by using the mass spectral data to search both a tomato protein and a decoy database (ftp.sgn.cornell.edu/proteins) using Mascot version 2.2.
We show that this strategy not only provides information about the complement of tomato fruit extracellular proteins, but also constitutes an excellent approach to isolate apoplastic/cell-wall localized proteins with minimal contamination by cytosolic proteins. We are using this methodology in conjunction with iTRAQ labeling for relative quantification of protein expression to study the tomato fruit secretome dynamics during fruit ripening.
Many scientific centers and life-science facilities today have access to medium to large computer clusters or large numbers of computers that can be employed in a grid-type computing environment. Often these clusters or grids employ some sort of batch queuing and job management system. X!Tandem, an open-source protein identification program, has been adapted to run on these environments by using Sun Grid Engine (SGE) as its job management system for submitting X!Tandem search jobs. Both X!Tandem and SGE are open-source platforms, allowing life scientists to fully utilize their computing resources without the significant investment typically required by proprietary solutions. Here, we describe an easy to use Web interface that allows X!Tandem jobs to be submitted to computer clusters for scalable computing. The results can be retrieved without any complicated interactions from the end user. The Web interface was modified based on thegpm project (www.thegpm.org). In addition, we have written the interface and backend applications to automatically create scaffold (www.proteomesoftware.com) files from the X!Tandem search output using the commercial program Scaffold Batch. All of the software written for this project will be made available under an open-source license.
High mobility group box 1 protein (HMGB1), a non-histone chromosomal protein, functions both as a proinflammatory cytokine and as a nuclear transcriptional factor. Acting through the RAGE receptor pathway, HMGB1 is critical for dendritic cell activation and essential for naïve T-cell survival and expansion. Our proteomic studies identified nuclear HMGB1 in human peripheral blood eosinophils. Since HMGB1 has a prominent role in immunomodulatory processes of specific interest for eosinophil-associated conditions, we investigated the subcellular localization and cellular release of HMGB1 in activated eosinophils.
Freshly isolated eosinophils were stimulated with GM-CSF, IFN-γ, or TNFα for 3 and 24 h, followed by the isolation of subcellular fractions, IEF, SDS-PAGE, MALDI-TOF-TOF, and Western blotting.
In nonstimulated cells, nuclear HMGB1 levels were significant, whereas cytoplasmic levels were not; after cytokine stimulation for 3 h, cytoplasmic HMGB1 levels were elevated, indicating active nuclear-cytoplasmic translocation of HMGB1. GM-CSF and IFN-γ were more efficient in HMGB1 translocation induction than TNFα. After 24 h cytokine stimulation, nuclear HMGB1 levels remained high, whereas cytoplasmic HMGB1 levels were not significant. Analysis of immunoreactive HMGB1 after 24 h cytokine stimulation indicated extracellular HMGB1 release. In contrast, nonstimulated and apoptotic eosinophils did not release significant amounts of HMGB1 as assessed after 24 h.
Eosinophils express and actively release HMGB1 upon activation with GM-CSF, TNFα, or IFNγ. The ability of activated eosinophils to release HMGB1 supports the immunoregulatory role of eosinophil in the context of asthma and certain viral infections. We propose that strong immunoregulatory and proinflammatory properties of eosinophil-derived HMGB1 provide novel and intriguing mechanisms of eosinophil-associated pathologies.
This study was supported by the NIH/NHLBI Proteomics Initiative NO1-HV-28184 (AK) and NIH NCI 1R24/CA88317 (AK).
The effect of high temperature during grain fill on the accumulation of KCl-soluble/methanol-insoluble albumins and globulins was investigated in the endosperm of developing wheat (Triticum aestivum, L. cv. Butte 86) grain. Plants were grown under a moderate (24°C/17°C, day/night) or a high temperature regimen (37°C/28°C) imposed from 10 or 20 d post anthesis (dpa) until maturity, and heads were collected at selected time points during grain development. KCl-soluble/methanol-insoluble albumin and globulin proteins were isolated from the endosperm, separated by 2DE, and identified by LC-MS\MS. Developmental profiles based on 2DE gel spot intensity were derived for nearly 200 proteins and analyzed by hierarchal clustering. Comparison of protein profiles across physiologically equivalent stages of grain fill revealed that high temperature shortened, but did not substantially alter, this developmental program. Accumulation of proteins during development shifted from those active in biosynthesis and metabolism to those with roles in storage and protection against biotic and abiotic stresses. Few proteins responded transiently when plants were transferred to the high temperature regimens, but levels of a number of proteins were altered during late stages of grain development. Specific protein responses depended on whether the high temperature regimens were initiated early (anthesis or 10 dpa) or mid (15 or 20 dpa) development. Some of the heat-responsive proteins have been implicated in gas bubble stabilization in bread dough, and others are suspected food allergens.
Influenza vaccination is the primary method for preventing influenza and its severe complications. Licensed inactivated vaccines for seasonal or pandemic influenza are formulated to contain a preset amount of hemagglutinin (HA), the critical antigen to elicit protection. Current methods to establish the HA concentration of vaccines rely on indirect measurements subject to considerable experimental variability. We present a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the absolute quantification of viral proteins in a complex mixture. Using an isotope dilution approach, HA from viral subtypes H1, H3, H5, and B was determined both directly and rapidly. This method can be applied to purified virus preparations, monovalent bulk concentrates, or trivalent inactivated influenza vaccines with improved speed, sensitivity, precision, and accuracy. This LC/MS/MS approach may substantially increase seasonal vaccine production reliability and reduce time and effort to deliver a strain-specific influenza vaccine for public-health use during the next influenza pandemic.
In order to characterize in-vivo interactions in Drosophila melanogaster and further our understanding of developmental processes, triple-tagged YFP D. melanogaster traps with Flag and Strep affinity tags were generated to isolate multi-protein complexes in a high-throughput fashion. The incorporated Flag and Strep affinity tags allow the target protein to be isolated from its native environment, by parallel or tandem affinity purification, along with any associating proteins. Each resulting pulldown eluate is analyzed in a single run by LC-MS, and the bait and associating proteins are then identified using the Mascot search engine in conjunction with FlyBase.
At the data analysis level, we have utilized Proxeon’s ProteinCenter software to visually and statistically compare multiple parallel pulldown datasets and therefore identify only true binding partners with high confidence. By analyzing many lines together, we can determine common contaminants immediately as well as pulldown-specific contaminants. These data are used to compile a nonexhaustive exclusion list used in the data-dependent settings for the upstream MS/MS analysis and thus increase the performance of the LTQ Orbitrap. The Venn diagrams function allows us to compare hits identified in the different pull-down methods and observe the overlap. Also, the biological annotation feature is useful in confirming relevant in-vivo interactions.
These data will be a valuable catalog of the D. melanogaster proteome and will complement the in-vitro data generated from the yeast 2-hybrid interaction screens.
Proteolytic modification of components of the extracellular milieu by metalloproteinases plays an important role in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS family of proteases, which is related to angiogenesis, inflammation, and cancer. We describe a proteomic screening for ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. The secreted proteins from the two cell lines were modified with different isotopomers of the nonisobaric ICPL label, mixed, and quantitatively compared in a SDS-PAGE LC-MS/MS workflow. Seven proteins were identified as putative substrates of ADAMTS1 and their proteolytic degradation characterized. Two criteria have been used to identify putative substrates of the protease: Increased amounts of the proteolytic fragments and/or a decrease on the full-length protein substrate in the ADMTS1 overexpressed conditions, the light ICPL sample. Using these criteria, we could identify the following proteins as putative substrates of ADAMTS1: nidogen 1, showing a 110-kDa upregulated protolytic fragment with a regulation value of 3.1-fold, and nidogen 2, showing upregulated proteolytic fragments with 3.0-fold and 2.9-fold regulation values. Newly identified substrates include: calsyntenin 1, for which a fragment comprising the ectodomain is observed with a 2.0-fold regulation; the Alzheimer disease beta-amyloid beta protein, showing a 55-kDa fragment with a 2.1-fold increase; fibulin-1, for which two fragments of 55 kDa (3.2-fold increase) and 37 kDa (4.6-fold increase) are observed; nucleobindin-1, for which a 2.4-fold decrease in the full-length protein is observed, together with a 4.2 fold increase of a 25 kDa fragment, and insulin-like growth factor binding protein 2, showing a 2.1-fold decrease of the full-length protein and a 5.0-fold increase of a 10-kDa fragment.
Superparamagnetic microparticles are, due to the large area of their solid phase and the magnetic core, very useful for the purification of substances from biological fluids. Normally, the shell of these microparticles is comprised of organic polymers and needs a protein coating—e.g., albumin or streptavidin—to minimize unspecific protein binding and aggregation of the particles. These protein coatings are also required for a crosslinking of antibodies to the particle surface when performing immunopurification processes. Unfortunately, protein coatings are often unstable under harsh elution conditions like acids, organic solvents, or SDS, which are required for the disruption of the antibody-antigen binding during immunopurification.
To avoid these problems, we synthesized modified magnetic microparticles based on silica and coupled an antibody against prostate-specific antigen (mouse anti-PSA-IgG) covalently to the surface of the particles. These “antibody particles” were used for the immunopurification of PSA from seminal plasma. After the binding process, PSA was eluted under mild conditions using a high-salt buffer and bound subsequently to custom-made magnetic reversed-phase particles, thereby avoiding sample loss compared to time-consuming dialysis. Compared to other magnetic microparticles and purification methods, this procedure resulted in a rapid and efficient purification of PSA in an almost pure form.
The custom-made magnetic reversed-phase particles were also used for the purification of substances secreted in very low amounts from cancer cells into cell-culture supernatants. When combining this purification procedure with LC-MALDI-MS analysis, we observed unequivocal differences in the peptide patterns secreted by two mamma carcinoma cell lines of different malignant potential.
In conclusion, we have developed different magnetic microparticles, which are very suitable either for immunopurification processes and the simultaneous binding of low concentrated substances from cell-culture supernatants.
Src family kinases (SFKs) are nonreceptor tyrosine kinases that play important roles in transducing signals through phosphorylation of a broad range of downstream substrates. These kinases are critical in regulation of cellular signaling, and their activity is tightly regulated through different mechanisms. One of the mechanisms of Src activity control is the phosphorylation and dephosphorylation of a regulatory tyrosine at position 535 (Y535). Inactive n-Src is associated with phosphorylated tyrosine 535 (pY535) and dephosphorylated tyrosine 424 (Y424), whereas the dephosphorylation of Y535 and phosphorylation of pY424 are associated with the active state of n-Src.
Mutating Y535 to phenylalanine (n-Src/Y535F) resulted in the hyperphosphorylation of pY424 and an enhanced n-Src activity. Additional mutation at lysine 303 to arginine (n-Src/K303R/Y535F) abolished the phosphorylation of Y424 as well as the kinase activity of n-Src. The different phosphorylation states of the wild-type n-Src and its mutants were immunodetected. Multiplexing strategy approaches including two-dimensional fluorescence difference gel electrophoresis and mass spectrometry were employed to determine and quantify the relative pY424/pY535 populations in the wild-type and mutant n-Src.
Acinetobacter baumannii is a nonmotile, obligate aerobic Gram-negative bacterium that has emerged as an important nosocomial pathogen. A. baumannii was known to be susceptible to the majority of antibiotics in the 1970s but now 80% of all Acinetobacter clinical isolates have become resistant to almost all currently available antibacterial agents. Nosocomial infections induced by A. baumannii have been implicated in epidemic pneumonia, urinary tract infections, septicemia, and meningitis. However, little is known about the virulence or antibiotic resistance of A. baumannii on the proteome level. In this study, membrane proteome of A. baumannii DU202 was analyzed in response to tetracycline and imipemen. About 280 proteins were identified from the membrane fractions from the A. baumannii DU202 by 2DE, iTRAQ, and LC-MS technology. More than 50% of identified proteins were integral proteins having more than one transmembrane domain. Up-regulated or down-regulated proteins were suggested by quantitative proteome analysis.
The pathways involved in the processes of fruit development and ripening are unique to plants and vary between species. Developmental, physiological, anatomical, biochemical, and structural differences contribute to the operation of unique pathways, genes, and proteins. Many of these have been characterized in the tomato, a climacteric-ripening fruit; however, nonclimacteric ripening such as that in citrus, is poorly understood. Improvements in the understanding of nonclimacteric fruit stocks may yield benefits both for public health and agricultural economics.
This study aims to identify and quantify developmentally regulated proteins in two stages of oranges in the nonclimacteric ripening species Citrus sinensis; in an earlier, still-green, smaller, acidic orange; and in a later, larger, sweeter, juicier, less acidic orange. In this project, soluble fractions of both types of orange were extracted separately and purified for analysis.
Accurate LC-MS/MS data were acquired with a Finnigan LTQ-FT for the differentially expressed proteins in the two developmental stages, then label-free techniques were employed to quantify differences between proteins of the two subsets—label-free spectral counting and comparison of peak intensities using the Sieve program. Statistical analyses were conducted with Spotfire software. A citrus genomewide ESTs database and the NCBI-nr (green plants) database were used to identify proteins, which were subsequently classified according to their putative and assigned functions to known biosynthetic pathways. A number of proteins were found to differ between the two stages of orange, suggesting significant shifts in some pathways; these may suggest mechanisms for the metabolic changes affecting the quality of the fruit and resistance to disease. These studies may also reflect some synergism between different pathways during maturation of the fruit.
For decades, the field of differential expression proteomics has been capable of providing only relative quantitation information. A diverse array of technologies have been developed and applied to differential expression proteomic studies, from the initial 2D gels to modern LC/MS (isotope labeled and label free) methods. However, the biological impacts of these relative quantitation methods have been limited in that each experiment provides information relevant only to the specific test and controls samples analyzed in that experiment. The utility of any proteomics experiment would be greatly enhanced if it could provide both relative and absolute quantitation information regarding all of the proteins identified. This provides a more complete understanding of the biology, moreover, providing absolute quantitation information on an ’omic scale allows the results of the proteomics experiment to be translated to other experiments, performed under different biological conditions at different times and in different laboratories.
In 2005, Silva, et al. reported a method for absolute quantitation1 based on the empirically observed fact that the average LC/MS response for the three most intense tryptic peptides per mole of protein is constant (±10%) across a widely diverse array of proteins. Spiking a known amount of an internal standard protein into the sample permits the calculation of a universal signal response factor, if the LC/MS data acquisition method records accurate abundance information.
This work describes our initial efforts towards the creation of a proteomics database of absolute quantitation information for nuclear and cytoplasmic compartments of cancer cell lines. This database addresses breast cancer and non–small cell lung cancer cell lines, specifically including those whose genotypic differences lead to refractory responses to drug therapies. The LC/MS analyses are accomplished using alternating scans of hi-lo collision energies, providing quantitative and qualitative information in a single LC/MS run.
An often-cited advantage of MALDI-MS is the ability to archive and reuse sample plates after the initial analysis is complete. However, experience demonstrates that the peptide ion signals decay rapidly as the number of laser shots becomes large. Thus, the signal level obtainable from an archived sample plate is often too low to be of any practical value upon reuse. Here we report a simple approach that can be used to regenerate peptide ion signals from apparently exhausted MALDI samples by hydration (“waterboarding”). We have found that the average signal obtained from 1600 laser shots decayed exponentially over 30 consecutive runs to the point where only 2% remained. After hydration, the signals of the five peptides evaluated increased an average of 11-fold compared to their level before hydration. The benefits of hydration were also confirmed by using tryptic digests of proteins separated by a 2D gel. Hydration of freshly prepared sample plates did not enhance and generally reduced the signal level obtainable from virgin samples. Control experiments that involved incubating the exhausted sample plates under vacuum or in sealed chambers under air at atmospheric pressure for various amounts of times did not regenerate the signals. The method presented will find practical use in applications that involve protein discovery using MALDI-MS by allowing for an increase in both the number and confidence of the protein identifications.
The Luteoviridae cause the most economically important virus diseases of cereal crops worldwide. These RNA viruses are transmitted by specific aphid species, once the virus navigates cellular barriers in the aphid gut and accessory salivary glands. The aphid proteins that facilitate virus transmission are mostly unknown; however, their identification is critical to the development of agricultural practices that utilize gene targeting as a mechanism to control virus spread. The aphid Schizaphis graminum (Sg) is a model system for studying transmission because genotypes differ in their vectoring capacity. F2 progeny of two Sg genotypes—an efficient vector of two luteoviruses (BYDV-SGV and CYDV-RPV) and a nonvector—segregated both for their ability to transmit SGV and RPV and the transmission barrier, indicating that transmission of closely related virus isolates is controlled by different alleles at the gut and salivary tissues. The F2 populations are perfectly suited to identify tissue-specific proteins involved in virus transmission using a comparative proteomics approach. Total soluble proteomes from vector and nonvector genotypes were compared using 2D-DIGE. Four proteins were identified that were exclusively found in vectors, including both a luciferase and a cyclophilin B homolog. Both protein families are involved in cytosolic macromolecular transport. Co-immunoprecipitation with purified RPV confirmed their ability to bind virus, supporting the hypothesis that they are involved in virus transmission. Membrane associated proteins are also expected to contribute to selective virus transmission at both the gut and salivary tissues. Thus, multiple extraction techniques were developed. Comparison of different extracts from vector and nonvector genotypes has detected additional protein spots unique to both phenotypes. Combining phenotypic separation using genetics with proteome separation using DIGE and immunoprecipitation provides a powerful technique to correlate protein presence with a biological trait.
The 8-plex iTRAQ reagents enable isobaric, multiplexed peptide labeling for quantitative proteomic analysis by using amine-specific, stable isotope reagents in up to eight different samples simultaneously. In this study, we describe protein expression profiles for staurosporine-induced apoptosis in HCT116 cells using 8-plex iTRAQ reagents with the QSTAR Elite LC/MS/MS system. In addition, based on the protein results, we also correlate protein expression with the mRNA expression profiles generated using Taq-Man Gene Expression assay.
Lysates and total RNA were generated from 300 nM staurosporine at 1, 2, 4, 6, 8, 12, 16, and 24 h treated HCT116 cells and nontreated cells. Each lysate was digested and labeled with 8-plex iTRAQ reagents according to standard procedures. The combined peptide mixtures were separated by strong cation exchange chromatography.
From the 14 strong cation exchange fractions analyzed, 1276 proteins were identified with 95% protein confidence or higher (with an estimated 2.2% false-discovery rate) and quantified using ProteinPilot 2.0 software. The PANTHER Classification System information enabled searching for protein expression patterns based on molecular functions and biological processes. Many of the identified proteins were chaperones, cytoskeletal proteins, or involved in nucleic acid binding. The direction of changes in expression levels between proteins and mRNAs were not always in the same direction, possibly reflecting post-transcriptional control of protein expression.
Eight-plex iTRAQ reagents provide useful expression regulation information on staurosporine-induced apoptosis in HCT116 cells. The combined use of 8-plex iTRAQ reagents, ProteinPilot 2.0 software, and the QSTAR Elite LC/MS/MS system provide the robust identification and relative quantitation solution required for protein biomarker discovery.
The typical manifestations of viperid envenoming are hemorrhage and edema at the site of the injury. In the case of hemorrhage production, the snake venom metalloproteinases (SVMPs) have been shown to participate in this process by proteolytically degrading extracellular matrix and capillary basement membranes, which in part lead to the disruption of local capillary networks, hemorrhage, and edema. Over the past several years, our laboratories have reported the effects of SVMPs on tissues and cultured cells using techniques such as in-vitro hydrolysis of extracellular matrix proteins, surface plasmon resonance, solid-phase binding assay, gene expression analysis, and immunohistochemistry. All of these studies have provided considerable insight into the cellular mechanisms associated with SVMP-induced pathogenesis. More recently, we have focused on the application of proteomic approaches to assess the effects of SVMPs at the level of a tissue in a living organism. HF3, a P-III-class SVMP, is the most potent hemorrhagic toxin from Bothrops jararaca venom. The target proteins for proteolysis by HF3 in vivo were evaluated by 2D electrophoresis (2DE) and mass spectrometric analysis of differential spots. Moreover, skin proteins were also submitted to direct trypsin digestion and LC/MS/MS. Mice were injected with HF3, and after 2 h the dorsal skin was sectioned and proteins were extracted and evaluated by 2DE. HF3 was also injected in the thigh muscle of mice, and after 4 h blood was collected and plasma proteins were analyzed by 2DE. As expected, some of the results corroborate previous data generated using in-vitro assays; however, new, previously unobserved substrates have been found to be cleaved in vivo that further extend our understanding of the effect of SVMPs at the level of the tissue/organism. Our results provide insights into the mechanisms for the development of hemorrhage typically observed upon viperid envenoming.
Financial support: FAPESP.
Discovery of pathways within pathogens that are critical for the successful invasion of host organisms is an area of intense research effort by scientists in both public and corporate laboratories. Mass spectrometry–based proteomics is one powerful tool that is being used to identify the proteins and related pathways that pathogens employ during infections. Prior to analysis, samples must be appropriately prepared in order to allow for the characterization of the proteome of interest. The preparation of these proteins associated with the infectious and reproductive stages is a challenging task that involves isolation of the organisms from the host system, as many cannot be simply grown in pure culture. Additionally, analysis of the proteins from the host system and from both the pathogen and host system combined may each require differing sample preparation strategies. Described here is a summary of strategies employed to extract proteins from different pathogen-host systems, including Salmonella typhimurium, Salmonella typhi, Vaccinia virus, Monkeypox virus, Anaplasma phagocytophilium, and Ehrlichia chaffeensis. Using multiple sample preparation techniques, fractionation of resultant peptides, and optimizing for small sample sizes, we are able to localize the proteins of interest (i.e., membrane-bound vs. soluble). We then use an accurate mass and retention time strategy (AMT tag approach) to analyze samples of limited size. There are specific advantages of analyzing the separated host material, pathogen, and the pathogen within the host; these include trade-offs in sensitivity, specificity, and potential loss of context. One example is that analyzing the human Monkeypox virus within the infected host cells revealed three viral proteins not previously detected in the purified viral particles, but with fewer overall virus proteins identified. Such studies hold promise to realize novel treatments for many existing and emerging pathogenic diseases.
The mRNA of a truncated form of the catalytic γ subunit of phosphorylase kinase (PhK), normally found as an (αβγδ)4 hexadecameric protein complex, was recently reported to be present in human brain.1 Its in silico translation predicted a protein of 202 amino acids (residues 1–181 identical to their counterparts in full-length γ1-386, plus a unique 21-amino-acid C-terminal tail). Our current study shows the presence of truncated γ protein (γ1-181) in human brain by 2D-PAGE immunoblotting. We also found that γ1-181 can be phosphorylated in vitro with rat brain protein kinase C (PKC). In contrast, phosphate incorporation by PKC into γ1-300 was negligible, suggesting that the phosphorylation site for PKC is located within the unique C-terminus of γ1-181. Upon its phosphorylation, γ1-181 became able to phosphorylate a tetradecapeptide corresponding to PhK’s natural substrate; but phosphoproteomics analyses of various tissues for possible targets of γ1-181 has yet to reveal an in situ substrate. However, the γ1-181 did inhibit, PhK’s activity (apparent IC50 ~50 nM). This effect may be due to a PTSRPRVL stretch in γ1-181, which is also found in the regulatory protein ral2 and partially in the β subunit of PhK. The presence of γ1-181 in brain and the fact that it can be phosphorylated by rat brain PKC suggests a possible role for this new γ variant in signal transduction, perhaps through its inhibition of PhK and thus glycogenolysis. (Supported by NIH grants DK32953 and P20RR16481.)
Toxicology-based studies require a multidisciplinary approach to understand risks associated with contaminant exposure. While many use toxicogenomic tools, currently few use proteomic tools for biomarker discovery. The University of Florida has a well-equipped proteomic core with the newest mass spectrometers, including a hybrid quadrupole-TOF (ABI QSTAR XL) and hybrid quadrupole-linear ion trap (ABI 4000 QTRAP). We are using both large-scale and targeted proteomic techniques to evaluate the effects and health risks of contaminants that act as steroid mimics (estrogen (E2) and testosterone) mediated through receptors. We have used the iTRAQ method (Applied Biosystems) successfully to evaluate the effects of compounds that block or bind androgen receptors in liver of fathead minnows. We identified proteins involved in the oxidative stress response (e.g. catalalse, thioredoxin) and translational response (e.g., ribosome L23a, S8) as being differentially expressed after treatment to an androgenic compound. We compared this to gene expression and showed altered protein abundance correlated with mRNA levels in some cases (e.g., catalase) but not in others, suggesting complex regulation. We are also developing a multiple reaction monitoring (MRM) method using the quadrupole linear ion trap (QTRAP 4000, Applied Biosystems) to detect low-abundant E2 receptors (ERs) in complex mixtures. Recombinant ER proteins were first produced to develop the MRM as standards. We will use targeted peptides that are detected through MRM consistently as AQUA internal standard peptides synthesized by the facility. We also have produced recombinant proteins for specific regions of the ERs to produce polyclonal antibodies for the three subtypes in collaboration with the ICBR Hybridoma laboratory. We demonstrate the utility of using both genomic and proteomic tools in identifying candidate biomarkers of chemical exposure. Research supported by NSERC postdoctoral fellowship and the Superfund Basic Research Program from the National Institute of Environmental Health Sciences P42 ES 07375 and RO1 ES015449.
Mass spectrometry has become a major analytical tool for protein identification in complex biological samples. One of the main workflows for shotgun proteomics is an initial separation of proteins by gel electrophoresis followed by their digestion with trypsin, further separation of resulting peptides by reversed-phase HPLC, and mass spectrometry analysis. This procedure combines the very high resolving power of electrophoresis and HPLC for proteins and peptides respectively, with the high sensitivity of mass spectrometry. However, high sensitivity of mass spectrometry is often compromised by low sequence coverage of proteins due to a large number of short tryptic peptides. One possible way to increase sequence coverage for proteins is to increase average tryptic peptide length by a modification of lysine residues. An increase in protonation efficiency often observed in peptides containing modified lysine residue can also be beneficial for a mass-spectrometry analysis.
In this study we developed a protocol for automated modification of lysine residues using the ProGest apparatus (Genomic Solutions, Inc.) to explore the feasibility of improving the sequence coverage of proteins. According to this protocol, proteins in gel slices were modified with NHS-biotin followed by trypsin digest and extraction of peptides from the gel. Peptide mixes originated from labeled and control proteins were analyzed by MALDI-TOF-TOF.
Our data indicate that nearly half of the lysine residues were labeled, with efficiency ranging from a few to nearly 50%. The lysine residue labeling resulted in an increase in the number of peptide assignments and an improvement of the sequence coverage of tested proteins. We conclude that this method of automated labeling of lysine residues using ProGest can be used in sample preparation for mass spectrometry to improve the overall success rate of protein identification in complex mixtures by increasing the protein sequence coverage
Ingestion of blood by the yellow fever mosquito Aedes aegypti triggers the release of the diuretic peptide aedeskinin, setting off a cascade in Malpighian tubules (kidneys) culminating in increased fluid secretion rates (diuresis). Aedeskinin is known to bind to a G protein-coupled receptor and to raise intracellular Ca2+ concentration. Little is known about the cytoplasmic proteins involved in the mechanism of action of aedeskinin. The goal of this work was to use 2D gel-based proteomics analysis for cytoplasmic proteins in control Malpighian tubules and in tubules treated with aedeskinin (10−7 M).
More than 2300 Malpighian tubules were isolated for each control and aedeskinin-treated (2 min) group. The tubules were homogenized, and the cytosolic proteins were obtained by ultracentrifugation. The proteins were separated by 2D electrophoresis using 24-cm, pH 3–10 nonlinear IPG strips in duplicate. All gels were stained with Pro-Q diamond, imaged, and further stained with SyproRuby. All images were analyzed by ImageMaster 6.0. Image analysis identified 127 spots whose expression varied by more than a factor of 1.5 between control and aedeskinin groups, and 70 spots visualized with Pro-Q stain. A total of 177 spots were picked for nano-LC-ESI-MS/MS analysis. The MS/MS data were submitted to Mascot search engine against a normal and decoy Aedes aegypti database downloaded from NCBInr.
The results showed that 162 out of 177 spots were successfully identified as 235 distinct proteins, with a false-discovery rate near 1%. The identification of 223 of 235 proteins was based on multiple-peptide hits, and 18 phosphoproteins were directly identified with MS/MS analysis without additional enrichment. The analysis of identified proteins by protein function classification and subcellular location using Blast2GO and PSORTb programs provides insights into the physiological roles of these proteins in mediating the post–blood meal diuresis in the mosquito. Supported by NIH R21 AI0702102.
The reliability of biomarker discovery by means of proteomics has been called into question. Inadequate sensitivity and reproducibility are among the chief concerns. The hamburger effect, proposed by Dr. Diamandis in an editorial by Dr. Garber, hypothesizes that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound biomarker research. Little is understood about the variability of complex proteomes in response to the environment.
Two methods, LCMS and 2DGE, were used to study the hamburger effect in two cross-sections of the soluble fruit fly proteome. 2DGE measured abundant proteins, whereas LCMS measured small proteins and peptides. Proteomic differences between males and females were first measured to demonstrate the discriminatory capability of the methods. Ethanol was added to the diet of some populations and no significant differences were reported. Differences were observed by LCMS after a 24-h period of starvation. Three of about one thousand molecular species were altered significantly in the starved populations, suggesting that the influence of even an extreme diet change on the variability of the proteome or peptidome is modest, and that the abundant proteome of the whole organism is robust.
We studied patients with familial adenomatous polyposis (FAP), because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts, and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis and mass spectrometry. Approximately 13% of 1695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers. Many of the expression changes affect pathways consistent with the function of the APC protein, including cell adhesion, cytoskeletal organization and biogenesis, cell motility, mitosis, apoptosis, and transcription. Prominent changes observed point to attenuation of apoptosis and are compounded by elevation of many mitochondrial enzymes, proteins associated with energy metabolism, and oxidative stress response.
A common technique to analyze proteins by mass spectrometry is to digest the proteins into peptides with an enzyme, separate the peptides by reversed-phase chromatography, and introduce the peptides into a mass spectrometer by electrospray ionization (ESI). The enzyme most widely utilized for the digest is trypsin. The obtained peptides ionize via ESI to low-charge states, which are optimal for collision-induced dissociation (CID) mass spectrometry. While CID MS/MS is a very powerful technique, it does impose some limitations.
Electron transfer dissociation (ETD) is a new method to fragment peptides that utilizes ion/ion chemistry. ETD fragments peptides by transferring an electron from a radical anion to a protonated peptide. This induces fragmentation of the peptide backbone, causing cleavage of the Cα-N bond. This creates complementary c- and z-type ions instead of the typical b- and y-type ions observed in CID. The best ETD spectra are obtained from peptides detected at m/z 300–900 with a charge state of 3 or greater.
Here, we present the utility of ETD mass spectrometry for sequence analysis of the proteome of Arabidopsis thaliana.
The availability of both a complete genome sequence and high-quality genome annotation makes Arabidopsis an ideal system to develop new proteomics technologies.
Due to the complexity of a dynamic proteome, different approaches have to be combined to measure protein expression and dynamics, stress and developmental responses, post-translational protein modifications and protein interaction. So far, about 700 proteins from isolated Arabidopsis thaliana chloroplasts have been identified by mass spectrometry, whereas the predicted number of proteins targeted to the plastid is approximately 3000. Here, we present a successful approach for increasing the number of identified proteins of a serine protease-digested whole soluble fraction as well as gel-separated subfractions from isolated chloroplast stroma.
Unbiased biomarker discovery workflows for plasma and serum have mostly focused on the identification of differentially expressed candidate proteins and their tryptic peptides. This shotgun approach is severely limited by the dynamic range and diversity of proteins in blood. To ameliorate this problem, many protocols require the depletion of abundant proteins such as albumin and others by various affinity methods. A major caveat is that endogenous (nontryptic) peptides are typically tightly bound to albumin and other high-abundance carrier proteins in vivo, and are therefore lost in the depleted sample. Endogenous peptides are likely candidate biomarkers for many diseases and pathologies as they are secreted from tissues and enter the bloodstream. This phenomenon may explain why there has been little success in biomarker discovery using most shotgun methods. Endogenous peptide recovery from blood poses numerous hurdles. First, the proper collection and storage of blood samples minimizes the generation of artifactual peptides that may be generated ex vivo. Second, the large dynamic range in molecular sizes and abundance requires the separation of proteins from peptides and metabolites, and the separation must be done under denaturing conditions to ensure the recovery of endogenous peptides bound to carrier proteins. Last, because of the difficulties posed by the identification and sequencing of nontryptic peptides, very high resolution MS2 and MS3 data must be acquired in a reproducible and robust manner. In this study, we describe the development of a work-flow specifically geared toward the efficient recovery and identification of endogenous peptides from blood plasma and urine utilizing a combination of up-front batch sample prep and on-line liquid chromatography coupled with high-resolution tandem MS using HCD and CID fragmentation on an Orbitrap XL. Quantitative differential analysis of the endogenous peptides was carried out using label-free analysis with SIEVE software algorithm.
Part of the challenge of biological experimentation is the identification of technical influences on your results. Even when employing statistical procedures to highlight and rank significant up- or down-regulation of proteins, it is important to investigate the potential influence of such effects that may have been introduced during the preparation of the samples.
We will present a method for the in-depth investigation of sources of experimental variation, enabling extraction of biologically relevant results, in addition to highlighting areas of non-biologically influenced protein changes.
We use a statistically led approach to ask different questions of the data and so separate those protein changes that are related to, for example, gel, dye, or other technical factors from the biology. This allows the investigator to build confidence that the observed changes are of actual biological relevance.
In current implementations of SEQUEST, MS/MS spectra are pre-processed to reduce the effects of noise when performing peptide identification and scoring. Precursor peptide fragments often appear as overwhelmingly dominant peaks in the spectra, and to compensate for this, standard SEQUEST performs a two-pass normalization—first across the entire spectrum, and then within arbitrarily sized windows. To reduce low-intensity noise, the spectrum is subjected to a thresholding and smoothing algorithm derived from the fast Fourier transform.
Unfortunately, this spectrum pre-processing fails to take into account high-sensitivity MS/MS instruments like Orbitrap-FTs. These types of instruments produce data with dramatically different intensity profiles; the precursor fragment peaks are generally lower, and the ambient noise from the instrument generally higher. In SEQUEST cross-correlation scoring, this has the effect of causing otherwise poor peptide identifications to be scored highly, reducing confidence in the results.
We propose a third pre-processing step that aims to improve score differentiation for data originating from high-accuracy instruments. Prior to SEQUEST pre-processing, a spectrum is intersected with a theoretical peptide, using an intersection tolerance that is instrument dependent. This overlap spectrum is then sent into the standard SEQUEST flow, undergoing normalization, thresholding, and scoring. By removing any undesirable noise peaks, their effects on SEQUEST pre-processing are non-existent, allowing SEQUEST to score them like clean spectra.
Applying this scheme to data generated from an Orbitrap-FT analysis of a multiprotein sample mixture shows improved differentiation between good and poor peptide identifications, thus validating the strategy.
A major challenge in proteomic studies is the elimination of false-positive identifications. One method for minimizing false positives is searching a database containing reversed sequences;1 however, this method appears to underestimate false positives.2 The number of false positives in lists of identified proteins can quickly be judged by comparing the molecular weight (MW) distribution of identified proteins to the distribution from the protein database. The average MW of proteins in the human database is 52 kDa (median 40 kDa) and the distribution is very similar across species. Replication is another tool that is appropriate to use in proteomics to assess data quality. True protein identifications should repeat in replicate injections of a sample, whereas false-positive identifications should not. Searching three replicate injections with a 5% false-positive rate can lead to a greater rate (as much as 15%) if all three results are collapsed into one list without taking replication into account.
Protein identifications from a variety of samples were made by acquiring data with alternate scanning (MSE) followed by a database search that uses physiochemical properties to find true positive identifications. As more proteins were identified, the identified protein distribution matched the database more closely. To mimic bad proteomic data, a random protein dataset was generated by picking 5000 peptides out of a database at random. These 5000 peptides pointed to 3800 proteins, and 37% of the “identifications” replicated each time the experiment was repeated. For a true proteomics experiment, the replication should be obviously greater than 37%. The average protein MW of the randomly identified proteins was 72 kDa. Many of these protein “identifications” were to the highest MW proteins in the database, such as titan.
Conventional protein biomarker discovery investigations are predominantly performed with samples such as serum and plasma. Although such investigations result in the identification of many candidate biomarkers, seldom do such biomarkers find a place in the clinic, in part due to low sensitivities and specificities for disease diagnosis. In many cases this low diagnostic power is reflective of their origin; for example, inflammation markers for cancer, which indicate the presence of cancer but do not accurately indicate the site of malignancy. While such markers may be useful to indicate the presence of disease, they are not often sufficiently sensitive or specific to provide for clinically useful diagnoses.
We have developed a detailed workflow that employs FT-MS and label-free quantification for biomarker investigations from tissue interstitial fluid (TIF) harvested from the kidney tumor microenvironment. Tumor tissue and normal, adjacent tissue were dissected from radical nephrectomies less than 10 min after being removed from the cancer patient, diced into 1 mm3 sections, placed in phosphate-buffered saline, and allowed to incubate for 1 h at 37°C in a 5% CO2 atmosphere. The supernatants were collected and equal amounts of proteins were digested from both the normal and tumor TIF. The TIF digests were analyzed by liquid chromatography coupled online with tandem MS on an Orbitrap MS, and processed using SIEVE. So, far we have identified 871 differentially expressed proteins, of which 463 are decreased in abundance and 408 are increased in abundance in kidney cancer TIF. Several plasma proteins have been identified along with those involved in cell signaling, integrin signaling, cell-to-cell signaling, and cancer. Future directions include expanding the patient cohort, evaluation of the reproducibility of the workflow with the use of internal standards, and employing targeted SRM analysis of candidate tumor biomarkers.
Multidimensional LC-MS/MS-based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography (RPLC) in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension, as IEF and RPLC represent two orthogonal separation techniques. Free-flow electrophoresis (FFE) has the advantage of very high sample load, short separation times, and no regeneration time between consecutive runs, which is an attractive feature of the IEF dimension when doing shotgun proteomics experiments.
So far, either hydroxypropyl methyl cellulose–based or mannitol and urea–based separation media were used for the separation of peptides when performing FFE-IEF. With the modified protocol containing urea and mannitol in the separation media, sample processing before LC-MS/MS was improved significantly, resulting in fewer interfering peaks. In this study, we have optimized the FFE protocols and media further to improve resolution and compatibility with LC-MS/MS, and to comprise post-translationally modified peptides. Most peptides focus in the pH region from 3.5 to 5. A new FFE protocol was developed that doubled the number of FFE fractions in this pH region and increased the resolution accordingly to reduce under-sampling in the mass spectrometer. Urea-free separation media were introduced to facilitate coupling to LC-MS/ MS. No purification of the FFE fractions was required, which allowed direct introduction of the sample into the mass spectrometer and minimized losses through sample handling. Post-translationally modified peptides were isolated based on the differences in migration in the electrical field. The modified peptides were at the same time isolated but also separated to improve the identification rate from complex mixtures.
Embryonic stem (ES) cells derive from the inner cell mass of the blastocyst, an early-stage embryo. ES cells are pluripotent, being able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. They are therefore an outstanding model system for early development studies or for the comprehension of mechanisms of organ formation or organ regeneration studies in clinical research. Cripto is a growth factor involved in stem cell differentiation, and mouse cripto−/− ES cells have been developed and utilized to investigate their fate under a variety of conditions.
Here, a 2D LC-MS/MS approach has been used to qualitatively profile the cripto−/− embryonic stem cell proteome. The method is based on the multidimensional separation of a complex tryptic digest mixture using a nanoscale LC system online connected to an instrument capable of data-directed switching between the MS and the product ion MS/MS modes. The research enabled the recording of a large amount of data by loading low sample amounts. Protein identifications achieved via databank searching the ESI-MS/MS spectra provide qualitative information on the proteins present in the complex mixture. Furthermore, the identified proteins have been classified in terms of subcellular localization, molecular function, and biological process as defined by their associated Gene Ontology annotation. A quantitative profile of the cripto−/− embryonic stem cell proteome was obtained by performing a label-free quantitative LC-MS experiment, utilizing multiplexed (alternate scanning) LC-MSe. The principle of this method is based on measurement of the intensity of the peptides identified relative to their constituant proteins. Several differentially expressed proteins have been identified and quantified in cripto-deficient stem cells with respect to the control murine RI embryonic stem cells. Delineation of the relative amounts of these proteins can provide an integral view of the alterations induced in stem-cell functions by deleting the cripto gene.
Various proteins were resolved on ion-exchange columns—mixed-bed columns (PolyCAT A and PolyWAX LP) in the case of cell lysates and cation-exchange (Poly-CAT A) in the case of hemoglobin variants. Pressures of ~13,000 psi over the entire column resulted in elution 30% earlier in the gradient. High linear velocities and pressures of ~5000–6000 psi resulted in both a fourfold increase in the speed of separation and a simultaneous improvement in resolution in some cases. The effect is on selectivity; certain proteins were seen to shift in retention time relative to others. These effects may be traced to the increase in energy in the system conferred by high pressure, analogous to an increase in temperature, and possibly to differences in compressibility of different proteins. It was possible to resolve proteins from a cell lysate using a volatile mobile phase: an ammonium acetate gradient. This technique can be used as a rapid top-down separation step to distribute complex mixtures of proteins into fractions for identifications in proteomics.
LC/MS analysis of complex protein mixtures such as whole-cell digests or digests of biological fluids generally produce abundant protein identifications and some understanding of the relative amounts of each present. Insight into biological meaning or simply which experiment to do next requires extensive and specific information about each identified protein. Fortunately, public databases such as NCBI protein and UniprotKB/Swiss-Prot provide comprehensive descriptions of proteins, often providing clues for subsequent, more targeted experiments. However, without additional tools, the researcher has no alternative but to query these databases one protein at a time, a laborious and time-consuming process. The goal of this study was to establish annotation workflows for LC-MS/MS data of human cerebrospinal fluid (CSF) using a new set of tools. The annotation tool is integrated into a new MS data analysis package (Discoverer) that automatically retrieves pertinent information about each identified protein from public databases. Information retrieved by the workflows provides annotation including, but not limited to, GO (Gene Ontology) classifications, sites of post-translational modifications, PubMed references, and genomic information. Digests were prepared with several different proteases, and individually analyzed on a Finnigan LTQXL with ETD, either with CID alone or with a combination of CID and ETD to achieve superior sequence coverage and additional protein identifications. Peptide sequences were annotated using knowledge discovery environment–based workflow tools. Preliminary results indicate that the annotation capabilities presented provide specific information about proteins of the CSF and a convenient method to design iterative and targeted follow-up experiments.
Database search algorithms that are widely used with CID spectra, such as Mascot and SEQUEST, have recently been used by researchers with ETD spectra. However, ETD and CID generate spectra of completely different characteristics. The fact that ETD generates high-quality spectra for peptides of higher charge states forces researchers to carry out multiple searches for each ETD spectrum generated by a low-resolution system. A new algorithm, ZCore, specifically takes into account the unique characteristics of ETD spectra. It includes a data-preprocessing step that assigns charge state to precursors. ZCore is evaluated in comparison with Mascot and SEQUEST for identification of peptides with ETD spectra.
ETD data files were acquired on the LTQ XL ETD system. Search was carried out using standard SEQUEST, Mascot 2.0, and ZCore against Uniprot database in both forward and reverse directions.
The precursor charge state assignment function of an MS/MS data-preprocessing step was evaluated using our standard set of ETD spectra. The data-preprocessing step assigns a charge state to precursor ions according to unique ETD spectral characteristics. Ninety percent of the time, a single correct charge state was created, while 10% of the time, when confident assignment of a single precursor charge state was not possible, the two most likely charge states were assigned. The charge state assignment of ETD spectra reduced the number of spectra for a database search more than fivefold compared to ETD data analysis without preprocessing. For peptide identification from ETD spectra, ZCore demonstrates higher sensitivity and specificity than either SEQUEST or Mascot. ZCore also generates higher Cn scores. This larger score difference between the first and the second best hits indicates that this algorithm is more discriminatory than either SEQUEST or Mascot.
The goal of many proteomic experiments is the identification of proteins from complex biological mixtures. In many such experiments, particularly LC-MS/MS experiments, chromatography systems have been intentionally overloaded in the belief that a significant increase in the number of protein identifications will be obtained.
We have previously reported (ASMS 2007) that the number of proteins identified and extent of sequence coverage observed when a dilution series of an E. coli digest standard is analyzed by LC-MSE, an alternating low and elevated collision energy acquisition mode, and a novel ion accounting databank search algorithm, rises sharply with increasing sample loading until an optimum is reached (2.0 μg on a 150 μm i.d. column). Injection of further material gives a small increase of sequence coverage, but few additional protein identifications.
Here we will illustrate that as sample loading increases beyond the optimum for a given LC column diameter, non-ideal chromatographic behavior will begin to manifest itself. These effects include changes in chromatographic peak width, retention time, and non-linearity of ion detection. The latter effect in particular can degrade quantitative results.
The lists of proteins identified at each level of sample load have been carefully compared. The small set of proteins identified at the lowest sample load is consistently found at each higher loading, and are in fact the most abundant proteins in the organism. Moreover, the proteins identified at each loading level constitute a subset of those identified at the next highest level, with few “unique” identifications. This demonstrates that the data acquisition and processing produce the results that would be expected from serial dilution of the digest sample without producing false identifications from chemical noise in the dilute samples.
Additional samples will be studied.
Systems biology studies are gaining momentum, driven by success in genomics, transcription profiling, proteomics, and rapidly emerging metabolomic technologies. While powerful and sensitive techniques are available for the analysis of nucleic acids, proteins, and small molecules, major bottlenecks arise from the limitations of current sample-preparation techniques. Multiple samples are usually required because incompatible sample preparation methods are currently used to isolate distinct classes of molecules from cells and tissues. Moreover, the strong detergents and chaotropic agents commonly used to solubilize samples tend to interfere with subsequent separation and analyses. We have developed a detergent-free sample-preparation technique that allows concurrent isolation and fractionation of protein, DNA, RNA, and lipids from cells and tissues. This novel method relies on a synergistic combination of cell disruption by alternating hydrostatic pressure (pressure cycling technology—PCT) and optimized reagents that dissolve and partition distinct classes of molecules into separate fractions. We report rapid simultaneous isolation of DNA, RNA, proteins, and lipids from individual samples of cultured human fibroblasts, rat pheochromocytoma PC-12 cells, and several types of mammalian tissues. Gel electrophoresis and real-time PCR confirm that nearly quantitative recovery of intact genomic DNA and high yields of intact RNA are obtained using this novel technique. Additionally, high yields of proteins and lipids are obtained from the same sample for proteomic analysis. Total protein fractions obtained using this new technique have been analyzed by SDS-PAGE and two-dimensional gel electrophoresis. The results have been highly reproducible, and several protein species uniquely extracted by the new method have been identified by in-gel tryptic digestion and LC-MS/MS. Excellent reproducibility of the new method and high recovery of total protein, DNA, and RNA from several types of samples are demonstrated.
Alzheimer’s disease (AD) is the most common cause of dementia within the senior population and affects the part of the brain that controls thought, memory, and language. Nerve cells die in areas of the brain that are vital to memory and other mental abilities, and connections between nerve cells are disrupted. About 5% of men and women ages 65–74 have AD, and this may increase up to 50% at age 85. Currently, treatments exist that may be used to help slow down the progression of symptoms in people with mild to moderate AD, and therefore being able to diagnose the disease as early as possible is extremely important.
A biomarker discovery approach was undertaken using isobaric tagging technology (iTRAQ reagents) coupled to two-dimensional LC-MS/MS to identify potential bio-markers of Alzheimer’s disease from a set of cerebral spinal fluid patient samples (CSF). Fourteen CSF samples were obtained from both male and female patients with and without AD. The samples were affinity depleted for albumin and IgG prior to labeling, and then split into two experiments, one containing female disease and control samples and one containing male disease and control samples. Both experiments contained a global standard created by pooling both male and female control samples. The samples were then labeled with the iTRAQ reagents and pooled. In each case, this pooled sample was then fractionated by cation exchange chromatography and analyzed by LC-MS/MS on a MALDI TOF/TOF system. A novel software program was used to merge both datasets together, creating a single dataset of all 14 samples. Using multiple statistical packages, identified proteins were screened for differential expression proteins that may be potential bio-markers for Alzheimer’s. The results of both of these experiments will be discussed in detail.
With the advent of genome-wide sequencing projects, large-scale proteomics studies became viable for more varieties of different species. As a result, modern proteomics has to face the challenge of cross-species data comparison. This is a time-consuming, and sometimes almost impossible, task. Currently, the field of proteomics lacks sophisticated software tools to perform comparisons of large-scale proteomics data derived from different species. Here, we present a new development with the software tool Protein-Center, which enables complex comparisons of proteomics datasets at several levels. For example, a comparison can be made at the homology level as defined by the level of sequence identity, by shared peptides and peptide-based extraction of unique entries, and as a peptide-sharing algorithm, with the added requirement that group members must also share the same Sim60 group (60% sequence homology).
Using HUPO Brain Proteome projects deposited in the PRIDE database, we investigated the efficiency of the cross-species comparison based on the homology, peptide evidence, and peptide-sharing algorithm based on Sim60 grouping. The results of human and mouse cross-species comparison as well the efficient data mining and categorization of large datasets of proteins will be presented.
The search for new and validated biomarkers is of particular interest in various clinical areas, such as oncology, neurology, toxicology, and pharmacology. One of the challenges in finding the right technology for biomarker research is to combine a statistically reasonable throughput with an in-depth proteome technology. Proteolytic events play a significant role in disease-related phenotypes. Therefore, biomarker discovery projects based on top-down profiling approaches appear highly attractive, as proteolytic degradation products remain unaffected during analysis.
We applied high-resolution HPLC-MALDI profiling in combination with sample pre-separation for the analysis of undigested peptides and proteins in biofluids. It is known that this technology provides a high number of detectable peptides/proteins from, e.g., human serum (approx. 1500). In addition, LC-MALDI profiling is very reproducible in our setup as we use CCA thin-layer preparations.
In this feasibility study, human serum samples were used as a complex background matrix and spiked with different amounts of synthetic peptides to simulate biomarkers. Multiple LC-MALDI runs were analyzed by multivariate statistics (PCA) to identify the mock biomarkers. The accurate differentiation between groups of LC profiles as a function of the mock biomarker concentration was achieved. The LC profiles were archived up to several months on the MALDI PAC targets (plastic targets with prespotted CCA thin layers), allowing the complete data evaluation prior to peptide/protein identification of marker candidates. Therefore, the identification of detected markers was entirely decoupled in time, and MS/MS acquisition workload and disk space usage were drastically reduced. Direct MS/MS of smaller peptides or in situ digestion of larger peptides not amenable to direct MS/MS, provided for protein identification straight from the LC runs immobilized on the MALDI targets. Thus we narrowed the gap between detection of biomarker candidates and their final identification. Biomarker ID is mandatory for their proper validation and their further diagnostic use.
The fractionation of complex proteomic samples is critical to successful protein characterization and quantitation using mass spectrometry. Such samples often consist of tens or hundreds of thousands of peptides over a very wide dynamic range. Currently, the most widely used method of peptide fractionation is strong cation exchange (SCX) chromatography followed by reversed-phase separation of each SCX fraction prior to MS analysis. However, SCX exhibits less than optimal peptide resolution, resulting in the same peptide appearing in many fractions. Additionally, SCX fractionation typically results in a very uneven (front loaded) distribution of peptides among the fractions. These issues reduce the achieved chromatographic separation, increasing the redundant analysis of the peptides and further complicating the mass spectrum. A different first-dimension separation that does not exhibit such problems is therefore highly desirable.
We investigated the use of solution-phase pI separation as the first dimension of a 2D workflow and compared it with the standard SCX/reversed-phase workflow for a complex cell lysate spiked with standard proteins at various concentrations. The comparison focused on peptide distribution across fractions, number of proteins/peptides identified, and quantitation accuracy. These results will be described in detail.
A rapid AP-MALDI MS/MS-based proteomics protocol was developed to detect and identify Neisseria sp.–specific peptide biomarkers. Heat-inactivated clinical isolate cell suspensions belonging to Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to whole-cell on-probe tryptic digestion proteomic analysis. Amino acid sequences derived from three observed protonated peptide masses of m/z 1743.7, 1894.8, and 1946.9 were assigned to the corresponding proteins as Neisserial acyl carrier protein, conserved hypothetical protein, and putative DNA-binding protein, respectively, by atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry followed by selected ion tandem mass spectrometry and Mascot proteome database search analysis.
Accurate and quick identification as well as taxonomical classification of microorganisms are of interest mainly in research and in routine clinical diagnostics, which often is based on biochemical tests (API, Vitek technology). However, results of these tests are obtained after long incubation times, and costs per assay are significant. We report the application of an MS-based technique for the explicit differentiation of bacteria on the species as well as on the subspecies level.
Bacteria from a colony were applied to a sample target plate directly or after a short extraction protocol. After air drying and addition of matrix (CHCA), the samples were analyzed using a microflex MALDI-TOF MS (Bruker Daltonik GmbH). Specimens could be prepared in a few minutes from the plate, and a spectrum could be acquired within one minute. Spectra, consisting of a characteristic peak pattern, were mainly derived from ribosomal proteins, enhancing the robustness of the assay. Applying a dedicated software solution, the MALDI BioTyper, mass spectra for isolated microorganisms showed characteristic peaks. The latter could be used for correct identification by using a database (Bruker). It has to be pointed out that for successful subspecies identification, a consistent standardization of experimental procedures including culturing conditions should be applied.
Using sophisticated bioinformatics, not only ID on a species level but also a reliable subspecies recognition is possible.
The BioTyper-derived IDs outrange traditional biochemically derived IDs by subspecies capabilities. In addition, this technique allows for high-throughput screening at a fraction of the costs of consumables of traditional biochemical tests. Using MALDI-TOF mass spectrometry, powerful pattern-matching software, and a database of spectra of well-characterized microorganisms, a fast, robust, cheap, but reliable identification is possible.
Microwave energy is a powerful adjunct for enhancing the enzymatic digestion of proteins for proteomic analysis. Higher efficiency digestion is obtained for trypsin in 15 min using microwaves compared to conventional overnight digestion at 37°C. This is reflected in higher database search score results as well as higher intensity signals. The method has been applied successfully with solution and in-gel samples and is compatible with a range of enzymes, including trypsin, Lys-C, and chymotrypsin. Our latest results as well as an in-depth analysis into the actual basis of the enhanced search scores are presented in detail.
Proteomics profiling of complex biological mixtures using a nonbiased LC MS/MS strategy, Identity, and MSe on a QTof mass spectrometer, produces a list of candidate proteins whose differential expression ratio changes between control and disease state. Specific peptides from these proteins can be targeted as a marker for that protein in a screening assay using a triple-quadrupole mass spectrometer operating in the multiple reaction monitoring (MRM) mode. The MRM method, which is robust and reliable, is used to detect specific ions from target molecules and has recently received considerable attention in this area, where the simultaneous quantitation of large numbers of low-abundance proteins needs to be performed. In this mode of analysis, the sensitivity and dynamic range are improved and, providing sufficient data points across a chromatographic peak are recorded, quantitation is accurate. This high sensitivity coupled with the specificity/ selectivity afforded by MRM transitions allows extensive panels of peptide biomarkers to be monitored in a single experiment from complex mixtures.
The sensitivity of MRM analysis is further improved by coupling nanoscale UPLC to the mass spectrometer, which delivers separated components at high peak concentrations to the nano-electrospray ionization source with reproducible retention times. Appropriate MRM transitions and acquisition parameters, including accurate retention time windows, cone voltage, and collision energies, were determined from the Identity experiments.
In this study we have obtained a proteomic profile of protein expression changes using label-free LC-MSe and IdentityE processing of rat microsomal cells following perturbation with a range of chemical inducers, and from there determined a panel of target peptides for MRM quantitation studies.
Recent improvements in ESI-QTOF instrumentation make a novel MS-based approach to label-free protein quantification feasible, which will be a significant complement to current quantification methods, such as 2D gel–, label-, or MRM-based approaches. The scope of this presentation is an evaluation of accurate mass LC/MS for label-free quantification involving a nano-HPLC (Ultimate 3000, Dionex) connected to an ESI-QTOF (micrOTOF-Q, Bruker Daltonics). Our approach allows hypothesis-driven proteome analysis and discovery of potential markers resulting from supervised statistical analysis. While quantification is obtained purely from MS data, identification is done as subsequent step involving a targeted approach to obtain sequence information of relevant peptides and proteins from MS/MS data. Using newly developed software, we analyzed the reproducibility of peptide signals between different LC runs with respect to occurrence, retention time, chromatographic peak width, mass accuracy, and peak intensity or area. As a suitable sample we choose human lung carcinoma cell line A549 before and after TGF beta induction or spiked with a set of 12 standard proteins. For quantification, we used two samples, and for each standard protein the concentration ratio between the two samples was varied. In a targeted approach, using the peptide masses from a theoretical digest of the known standard proteins, we extracted accurate mass compound data from the LC-MS datasets. Suitable bioinformatics tools enabled us to match the compounds within replicate analysis of the samples. Initial results show that statistically valid quantification results are obtained with our instrumentation when performing a number of replicate analyses. The study covers the current status, the limitations, and chances of MS-based label-free quantification, and shows that this instrument will be an excellent tool for this application. The goals of the ongoing work are identification and validation of potential biomarkers from clinical studies.
It can often be difficult for individual facilities to assess their protocols and technologies with other labs. Recent work has shown that by following standard protocols, we can combne the data from 2D-gel experiments at the raw data level. This gives us the opportunity to take within- and inter-lab QC of proteomics experiments forward and allow an objective assessment of proteomics data to be produced by individual labs.
In a HUPO IAB study, it has been shown that, using defined running protocols and Progenesis SameSpots, cross-lab proteomics data can be combined at the raw gel level. An extension of this work allows us to take a fixed reference and allow a user to align their test gel to the reference. A fixed reference spot pattern is then applied to this gel, and a multivariate projection of the data (PCA) allows a very quick assessment of where this gel lies with respect to the reference set (and hence the global community). This procedure can be used across and within labs to not only test performance overall but also to assess any drift within a lab over time.
The work presented shows examples of data combined from multiple labs that have been through this workflow, and also provides a call to interested labs who would like to participate in building a suitable reference set. This set will be created with the intent of providing a resource for the community and Nonlinear will supply the capability for any lab to use these reference data free of charge and also to compare a reference sample generated in their lab against it.
Measurement of antibody production is important in vaccine design. However, the production, persistence, and heterogeneity of memory B cells may be more informative for long-term humoral protection against pathogens. Individual antigen-binding B cells participating in germinal center (GC) and post-GC responses were isolated via FACS. Subsequent single-cell genophenotyping revealed that responding B cells expressing diverse IgH genes can be subdivided into those being selected versus those being eliminated. Some memory B cells persist 4–5 mo following antigen encounter, suggesting value in vaccine design.
Real-time PCR (qPCR) remains one of the most sensitive and quantitative tools for gene expression analysis currently on the market. Although qPCR provides numerous opportunities for exciting discoveries, this technology is not without its difficulties. Two major hurdles in obtaining quality results from qPCR are (1) the design of both the assay and primer-probe sets used for detecting the gene of interest and (2) the interpretation of the data. At SeqWright, we help the researcher to overcome these hurdles through a variety of service offerings, including copy number determination of DNA or RNA targets, allelic discrimination/ SNP genotyping, microarray validation, microRNA analysis, biodistribution studies of gene therapy products, microbial detection and residual DNA analysis, and transgenic mouse genotyping. Our experienced staff can provide as much assistance as needed, from the initial study design to the final interpretation and statistical analysis of data. We also have the experience and infrastructure to perform FDA-level and research-level projects. At a time when qPCR holds the key to many exciting discoveries, SeqWright is committed to helping you open the door.
The BioMark 48.48 Dynamic Array enables running up to 48 real-time PCR assays on up to 48 samples; however, biomarker studies may require running more than 48 RNA expression assays to validate which are clinically useful. In order to maximize the number of assays that may be run in parallel on the 48.48 array, we have developed a procedure that involves combinatorial mixing of forward and reverse primers in order to generate functional PCR assays. Forward primers are added along one dimension of the 48.48 array and reverse primers are added along the orthogonal dimension. For example, in an 8 × 8 assay matrix, each row contains 8 different forward primers (e.g., F1, 2, 3, 4, 5, 6, 7, 8 in row 1) and each column contains 8 reverse primers (e.g., R1, 9, 17, 25, 33, 41, 49, 57 in column 1). After combinatorial mixing, any individual reaction chamber contains 16 primers, but the assay matrix is designed so that only two of these primers, one forward and one reverse, produce a PCR product. A different pair of primers generates a PCR product in each of the 64 reaction chambers of the 8 × 8 matrix, resulting in 64 distinct assays. Samples are added with one of the primer sets, for example, with the forward primers. Taken to the extreme, this method enables running 2304 assays on one sample using a single 48.48 array. Other combinations of 2304 reactions, such as 4 samples × 576 assays or 12 samples × 192 assays, are also possible.
The exceptionally sensitive nature of quantitative reverse transcription PCR (qRT-PCR) requires a set of rigorous controls to ensure the accurate interpretation of results. Many users unknowingly omit crucial validating steps, which may affect the quality of their data and confound the analysis. Optimal real-time RT-PCR reproducibility and reliability require: (1) minimal genomic DNA contamination; (2) intact RNA free of any impurities that inhibit the reverse transcription and PCR processes; and (3) correct PCR cycling conditions. We have developed a panel of control assays that ensure the quality of input RNA samples and monitor the performance of reverse transcription and PCR. They can easily be incorporated into any qRT-PCR experiment to provide quality-control checkpoints at each step. These assays consist of a genomic DNA contamination control, primers detecting a specific genomic DNA sequence within an ORF-free intergenic region; a reverse transcription control, primers detecting an external RNA control sequence spiked into the reverse transcription reaction; and a positive PCR control, an assay including an external DNA template and primers detecting it, producing a defined threshold cycle value under the correct PCR cycling conditions. Using this control panel, we will demonstrate how various suboptimal conditions affect qRT-PCR results, and highlight the importance of proper qRT-PCR controls to obtain reliable gene expression analyses.
We have developed a new Fast SYBR Green Master Mix that is a fast, reliable, and cost-effective solution for real-time PCR applications. The Fast SYBR Green Master Mix enables PCR runs in less than 40 min without compromising performance (regular cycle requires 90 min). This new master mix utilizes an instant hot-start mechanism that allows convenient benchtop setup, and the polymerase is activated on the first cycle. The much-reduced run time is accomplished by the instant hot-start and shorter cycling time. The Fast SYBR Green Master Mix sets a high level of performance in dynamic range, sensitivity, and copy number discrimination. The dynamic range is linear over 9 logs of template concentration. The sensitivity is detecting down to two copies of starting template and capable of discriminating 1.3-fold differences in template copy number. These performance attributes were accomplished while maintaining target specificity as well as benchtop stability. The new Fast SYBR Green Master Mix maximizes the throughput potential of fast-cycling-capable real-time instruments such as the 7900HT, 7500 Fast, and the StepOne series from Applied Biosystems.
The St. Jude Protein Production Facility (PPF) is a Cancer Center Support Grant–funded shared resource with the mission of stimulating the translation of discoveries in molecular and cellular biology to chemical and structural biology. The PPF has been in existence for seven years, has worked with over 50 investigators, and purified over 300 proteins. Expression services include primarily bacteria and insect cells, although yeast and mammalian cells can also be cultured. Protein purification is usually by affinity tag, with polishing achieved by gel filtration, ion exchange, or other column. Preliminary crystal screening trials are also offered, using prefilled 96-well trays. Molecular biology services are also available. Specialized equipment within the facility includes 10- and 120-L fermentors for bacterial cells; 8-L bioreactors and a Wave bioreactor for insect cell growth; a microfuidizer for cell disruption; and three AKTAs for protein purification. The PPF routinely and freely advises St. Jude investigators on all aspects of protein expression and purification, and is a central location for knowledge and equipment within the institution. The PPF interacts extensively with other shared resources, such as mass spectrometry, for quality assurance purposes on purified proteins. The PPF is also involved in other initiatives, such as production of vaccines, and in the development of production processes for protein therapeutics suitable for translation to a GMP.
The Protein Production and Characterization Facility of the Cornell University Life Sciences Core Laboratories Center provides an array of shared research resources and services to the university community and to outside investigators. The facility provides a concentration of advanced instrumentation and expertise in their applications. The goal of the facility is to meet the increasing need of investigators for purified proteins, molecular biology services, and instrumentation for kinetic studies and protein interaction characterization.
Baculovirus-mediated expression of recombinant proteins in insect cells is a valuable tool for producing soluble, active proteins. However, the traditional technique of generating recombinant baculovirus is time consuming and laborious. To accelerate the process of protein expression in insect cells, the InsectDirect System provides a rapid plasmid-mediated transfection approach that is ideal for generating small to moderate amounts of recombinant protein in 48 h, without creating recombinant baculovirus. For situations requiring a baculovirus approach, BacMagic DNA provides faster baculovirus production. BacMagic DNA eliminates the time-consuming and labor-intensive plaque purification steps, which were required to remove nonrecombinant parental virus. New pIEx/Bac vectors can be used for both rapid plasmid-based expression or to generate recombinant baculovirus, providing flexibility and greater optimization for insect cell expression. These dual-purpose vectors contain the Autographa californica nuclear polyhedrosis virus (AcNPV) enhancer/immediate early promoter combination (hr5/ie1) for plasmid-mediated and early baculovirus expression, and the AcNPV p10 very late promoter for robust baculovirus-mediated expression. To facilitate cloning, affinity purification, and subsequent proteolytic removal of affinity tags, 4 pIEx/Bac variations are available.
To assess vector performance, inserts encoding different types of proteins (an importin, a phosphatase, kinases, and Rluc) were cloned into pIEx/Bac vectors. Recombinant proteins were expressed by both methods, purified, and assayed. Results demonstrate that (1) for rapid, plasmid-based expression, pIEx/Bac vectors can be used to effectively screen clones and to produce small to moderate amounts of protein, and (2) for baculovirus expression, the pIEx/Bac vectors can be used to generate recombinant baculovirus for high-yield protein expression.
Expression screening is often needed in order to establish suitable conditions for difficult-to-express proteins. Frequently, multiple constructs of a specific target protein must also be analyzed. An affinity tag fused to the target protein in order to simply purification can also be used for enrichment during expression screening to improve detection. The purified protein can then easily be detected by SDS-PAGE, dot blots, or other techniques. The use of affinity chromatographic resin in multiwell plates for enrichment can dramatically increase the throughput, compared with standard column purification. In this study, His MultiTrap and GST MultiTrap, 96-well filter plates packed with Ni Sepharose High Performance and Glutathione Sepharose 4B, respectively, were used for the enrichment of tagged target proteins from crude or clarified cell extracts. The screening was automated with a Tecan liquid-handling station and a vacuum manifold. The expression levels were determined using SDS-PAGE.
Detailed characterization of recombinant proteins including the differentiation of isoforms or structural aberrations is a lot more difficult than the protein ID problem and its routine solution in proteomics workflows. Typically, a method mix is required that almost certainly involves protein separations, top-down (TD) plus bottom-up (BU) sequence characterization tools.
Two batches of a recombinant protein preparation were analyzed. They were characterized by MALDI-TOF, LC-MALDI-TOF, and TD and BU sequencing of the separated proteins. All sequences were analyzed with the BioTools 3.1 software (Bruker), which permitted dedicated TD sequencing combined with MS-BLAST (EMBL). Mascot 2.2 (Matrix Science) was used for all BU protein identifications and BioTools for the characterization of peptides that did not immediately match in database searches.
External a priori knowledge was used, such as the N-terminal sequence that was left from a thrombin cleavage site in the N-terminal His-tag.
Three different forms of advanced glycosylation end-product-specific receptor isoform were detected (MW range 12.5–33.5 kDa) in the two samples and characterized with regard to their differences using TD sequencing. Terminal truncation variants were assigned, and all forms were fully annotated to sequences from the NCBI-MR95clean protein sequence database plus the N-terminal sequence tag as defined in BioTools. TD and BU sequencing together with the TD-LC-MALDI analysis provided 100% sequence coverage of all three detected protein forms.
The human mitochondrial genome is composed of a small circular chromosome comprised of a total of 16 kilo-bases, encoding 37 genes. Owing to its maternal inheritance and high mutation rate, the mitochondrial genome can be employed as a molecular clock, and therefore can be exploited as a means to study the evolutionary history of populations. Damage and mutations in the mitochondrial genome can cause many diseases.
In order to create a high-throughput service to assist in the efficient study of the mitochondrial genome, a set of 24 primers were selected and adapted from the literature. These primers created overlapping fragments approx. 800–1000 bp with a sequence overlap between PCR segments of 150–200 bp. All primers were optimized to the same standard conditions. Each primer pair produced a clear, bright product band and clean sequence. The facility will make use of liquid-handling robotics to process high-throughput samples in 384-well format. Downstream analysis bioinformatics software is used to assemble, annotate, and genotype variants.
The recent introduction of new DNA sequencing technologies presents an exceptional opportunity for novel and creative applications with the potential for breakthrough discoveries. To support such research efforts, the Cornell University Life Sciences Core Laboratories Center has implemented the Illumina Solexa Genome Analyzer and the Roche 454 Genome Sequencer FLX platforms as academic core facility shared research resources. We have established sample-handling methods and informatics tools to build robust processing pipelines in support of these new technologies. Our DNA-sequencing and genotyping core laboratory provides sample preparation and data generation services and, in collaboration with the gene expression and informatics core facilities, provides both project consultation and analysis support for a wide range of possible applications, including whole genome assembly, amplicon resequencing, mutation detection, SNP genotyping, small RNA profiling, and genome-wide measurements of protein-nucleic interactions. Implementation of next-generation sequencing platforms as shared resources with multi-disciplinary core facility support enables cost-effective access and broad-based use of these emerging technologies.