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J Biomol Tech. 2009 February; 20(1): 31–89.
PMCID: PMC2670552

Poster Abstracts

J Biomol Tech. 2009 February; 20(1): 31.

SR1-S1 HRAS Mutations and 11p allelic Imbalance in a Rare Case of Agminated Spitz Nevus: Bridging the Gap between Bench and Bedside through Collaborative Research

Abstract

One of the major roles of the Nemours Biomolecular Core is to provide one-on-one mentoring for clinicians engaging for the first time in molecular genetics projects. This core function has successfully grown to include unique inter-institutional research collaborations. In this poster, we will present the detailed molecular analysis that was performed in collaboration with clinicians in DE and PA, on a rare and fascinating case of agminated Spitz nevus, and the methodologies developed for this project. Differentiating a Spitz nevus from a Spitzoid melanoma is often a challenging task faced by dermatopathologists. While the dysplastic nevus theory supports the notion that a Spitz nevus can progress to a melanoma, there is molecular evidence that refutes this possibility. Namely, Spitz nevi can carry unique mutations found in the mitogen-activated protein kinase (MAPK) pathway that are not found in melanomas. We used DNA sequencing as well as STR analysis for genotyping and copy number evaluation. The analysis revealed two HRAS “gain of function mutations” resulting in A11S and G13R amino acid substitutions in the ras protein and hyper activation of the MAPK signaling pathway. Allelic specific amplification PCR assays were developed to elucidate the allelic provenance of each mutation. We discovered that the maternally inherited allele carried both mutations in cis. The HRAS mutations observed in the patient’s Spitz nevi, HRAS copy number increase, as well as allelic imbalance at 11p with gain of the maternal allele, was also observed in tissue containing the mutated gene.

J Biomol Tech. 2009 February; 20(1): 31.

V1-S1 Improvements for Applying Single-Stranded Conformation Polymorphism (SSCP) Analysis on an Automated Medium-Throughput Capillary Electrophoresis System

Abstract

Single-stranded conformation polymorphism (SSCP) analysis is an established and proven method for screening and detection of single nucleotide polymorphisms (SNPs) and small deletions and insertions.

The SSCP analysis detects sequence variations through differences in electrophoretic mobility in a native gel system. A subtle sequence variant can cause a conformational change in the single-stranded DNA molecule that can result in detectable differences in mobility. Here we describe a formulation for a non-denaturing gel composition prepared from commercially available Conformation Analysis Polymer (CAP) in a glycerol and TBE buffer system. The polymer is used for capillary electrophoresis with the Applied Biosystems 3130/3130xl Genetic Analyzer along with size standard GeneScan™-600 LIZ®). New run modules were designed and are described that support the application on the instrument. We also present data that show how changes in temperature affect the outcome for screening for the H63D mutation in codon position 282 of the HFE gene.

The described conditions could be useful for SSCP-based applications like cost-effective screening for unknown SNPs in large number of samples.

J Biomol Tech. 2009 February; 20(1): 31–32.

V2-S1 The JAX Mouse Diversity Array: A New Very High-Density Genotyping Array

Abstract

The Mouse Diversity Genotyping Array is the most advanced high-density mouse genotyping microarray available. The custom Affymetrix array can assay over 600,000 single nucleotide polymorphisms (SNPs) and over 900,000 invariant genomic probes (IGPs). IGPs may be used to assay copy number variation. SNPs were carefully selected from publicly available SNPs to be highly polymorphic among characterized strains. There are approximately one SNP every 4.4 kb across the genome. 93% of all exons annotated by Ensembl have at least one IGP. All 238 mouse/human/rat ultra-conserved genomic regions have at least one IGP. All SNPs on the array have been validated with a prototype array. All SNPs and IGPs are annotated by position in the genome and by reference SNP cluster identifiers from dbSNP.

Example applications that can be performed by this array include:

  • Characterizing novel or existing mouse strains or lines—for applications such as association or QTL studies
  • Genetic quality control of strains
  • Characterizing or comparing any set of mouse DNA samples (e.g., tumor tissues or cell lines)—for applications such as array CGH

The technical performance of the array and computational methods have been evaluated by analysis of male and female DNA from a single inbred strain. A reference dataset of over 200 mouse genomes has been established which, as expected, increases the accuracy of genotype calls. Using these priors and optimized BRLMM-P parameters, the heterozygous SNP calls of inbred mice is significantly reduced.

J Biomol Tech. 2009 February; 20(1): 32.

SR2-S1 Methylation and Pathway Focused Gene Expression Analysis: New Core Services for Translational Research

Abstract

The mission of the Biomolecular Core lab is to facilitate discoveries that begin at a molecular level and move rapidly from research to the bedside. Methylation analysis and micro RNA studies are rapidly growing areas of interest in translational research. This poster will review the tools and approaches we used to set-up services for site specific and global methylation analysis as well as pathway focused gene expression and micro RNA studies. We will discuss the challenges and opportunities of establishing these new services in a clinical setting. We will describe how to design a site specific methylation assay in the study of mitochondrial DNA. Global methylation analysis, which is based on the use of methylation sensitive (HpaII) and methylation insensitive (MspI) enzymes, will also be presented. Pathway focused gene expression analysis was evaluated using SA Biosciences Human MAP Kinase Signaling Pathway RT2 Profiler PCR Array. These results were validated by both the ABI TaqMan assays and semi quantitative gel analysis. The micro RNA studies were performed using the Human miFinder RT2 miRNA PCR array kit with patient samples. We saw similar results in expression levels using both total RNA and enriched miRNA. Both the pathway and miRNA focused arrays perform consistently well in our hands and we highly recommend them as part of our Real-Time PCR service.

J Biomol Tech. 2009 February; 20(1): 32.

SR3-S1 Implementation of Fluidigm’s SlingShot Absolute Quantitation Product Offers the Dual Advantage of Replacing the Costly and Time-Consuming Titration Step as Well as Recovery of Sub-Optimal DNA Samples for 454 Next Generation Sequencing

Abstract

We have successfully implemented Fluidigm’s SlingShot technology into our 454 sequencing process at two key junctions: sub-optimal sample quantitation and replacement of the library titration. This implementation has allowed us to process previously failed samples (Roche recommends samples be between 3ug and 5ug) and replace the costly titration step saving us processing time (up to 2 days) plus the cost of a sequencing reaction.

Our initial experiment utilizing SlingShot consisted of 12 samples ranging in amounts from 990 ng to 2.5 μg. We processed these 12 samples using the approved Roche 454 protocol through library prep. After completion of the library prep, each sample was quantitated using the SlingShot product to accurately assess the amount of sample to go forward into bulk emulsion prep. Due to limitations of the existing quantitation methods available at the core genotyping facility (Ribogreen and Agilent), these samples would have never made it to the sequencer. We will present data highlighting the quality coverage attained (each sample had at least 20X coverage) from these samples using the quantitation obtained through SlingShot. We will also discuss future experimentation as well as future updates we would like to see with this platform.

J Biomol Tech. 2009 February; 20(1): 32–33.

V3-S1 Enhanced Workflow for Sequencing PCR Products by Capillary Electrophoresis

Abstract

Since the introduction of Sanger dideoxy sequencing, significant efforts have been directed toward increasing throughput by streamlining workflow. We describe here further enhancements for a PCR product resequencing workflow capable of reducing the total time, from beginning PCR reactions through completion of basecalling, to 6 hours or less. The workflow shown employs a new AmpliTaq Gold® Fast PCR Master Mix in conjunction with modified thermal cycler conditions to substantially reduce the time required for PCR amplification. Process time is further reduced through optimization of cycle sequencing conditions. We have coupled these improvements with an efficient sequencing reaction cleanup protocol and decreased Capillary Electrophoresis (CE) run time using MicroAmp® Fast 96-Well Reaction Plates on an Applied Biosystems 3130 Genetic Analyzer. Overall data quality compares favorably with data obtained from previously documented methods. The increased efficiency and generation of high quality results will reduce time to discovery in both clinical and research applications.

J Biomol Tech. 2009 February; 20(1): 33.

V4-S1 An Automated Microfluidic System for Integrated Thermocycling and Preparation of Sanger Sequencing Samples

Abstract

The poster describes the development of a sample preparation system for Sanger sequencing. The work covers the design of four integrated microfluidic chips, mated with a standard laboratory robot, to automate both dye-terminator sequencing reactions at the sub-1 μl scale and subsequent bead-based clean-up in glass microfluidic chips using SPRI beads. Data on the optimization of reactions for plasmid and PCR products are presented, including performance data for controls, and PCR and plasmid templates from core facilities. Microfluidic-scale reactions are contrasted to full volume control reactions for read length and signal-to-noise. Read lengths generated for plasmid samples range from 800 to 1,000 bp, using 125 to 600 ng/ul plasmid input concentration, and 0.2 to 1 ul of dye-terminator reagents. The poster also describes the basic function of the underlying microfluidic technology, which uses diaphragm pumps, actuated by pressure and vacuum, to distribute, mix and direct the flow of fluids and beads. This approach offers the prospect of lowering the cost of standard Sanger sequencing through reagent reduction and reduced ‘hands on’ time.

J Biomol Tech. 2009 February; 20(1): 33.

V5-S1 Optimizing Bisulfite DNA Conversion Method for Methylated CpG Island Discovery and Screening

Abstract

DNA methylation at the 5’ position of cytosine in CpG islands plays a critical role in the epigenetic regulation of gene expression. Typically methylation is inversely correlated with the transcription status of the gene.

Bisulfite DNA conversion is one of the most widely used techniques for methylation studies because of its relative simplicity, whereas other methods are frequently cumbersome and require significant optimization. The bisulfite conversion method allows precise analysis of methylation in a target region by converting all nonmethylated cytosines into uracils, while methylated cytosines remain unchanged. The workflows described here provide an effective solution for methylation analysis with straightforward protocols. We describe three different options: (1) clone-based sequencing, (2) direct PCR sequencing and (3) methylation-sensitive mobility shift assay. We also present a methylation work-flow decision tree that helps to direct to the appropriate method to choose for assessing the methylation status in the region of interest. These workflows are particularly useful for the analysis of samples where the amount of material is limited, and when analysis time is an important factor. Exemplary data from model gene systems are presented which validate the proposed workflows.

J Biomol Tech. 2009 February; 20(1): 33.

V6-S1 Applications of A New And Improved Size Standard For Fragment Analysis On Capillary Electrophoresis Instruments

Abstract

A good size standard is an essential tool for researchers in the field of plant and animal genomics for fragment analysis applications such as AFLP®, T-RFLP, VNTR, mutation screening, MLST, and BAC fingerprinting. We have developed a new high density size standard using Applied Biosystems’ 5th dye technology with a larger sizing range up to 1200 basepairs and no peak migration anomalies. Here we describe the Applied Biosystems GeneScan™ 1200 LIZ® Size Standard and illustrate its use with a variety of samples.

J Biomol Tech. 2009 February; 20(1): 33–34.

V7-S1 AutoDTR: Automatable, Reliable and Economic Sequencing Reaction Purification

Abstract

Most Sequencing Reaction Purification (SRP) methods require centrifugation which hinders the ability to effectively automate. Magnetic technologies offer a relatively lower cost and are automatable, but results are often variable with signal intensity fading along the electropherogram. Recently introduced ion exchange technology, requires extensive vortexing, use of special plate sealers, centrifugation and is expensive. We have developed a surface-modified micro-plate to perform SRP without the need for centrifugation or magnets. All manipulations are 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 the desired buffer. This new technology offers not only a robust, high-capacity, and fully automatable SRP platform, but also works efficiently with reduced volumes of BigDye® Terminators. Testing was done with a 96-well head on both a Biomek® FX and Hamilton MICROLAB® STAR Workstation in less than 30 minutes. A variety of conditions were interrogated, using high-copy and low-copy plasmid, PCR, Templiphi template and DNA Libraries. Typical Phred20 scores of 800 and complete elimination of dye-blobs was achieved with ABI BigDye® Terminator v3.1 volumes ranging from 250–1000 nL. With no adjustment to the protocol, the technology was very robust to variations in template and BigDye®. Thousand of sequencing reactions were purified with extremely reproducible results (CV 0.02–0.06) over a wide range of reaction conditions. This new technology offers a robust, high-capacity, and fully automatable dye terminator removal platform to work efficiently with reduced volumes of dye terminators.

J Biomol Tech. 2009 February; 20(1): 34.

V8-S1 Fast and Cheap DNA Sequencing Plasmid Template Preparation with an ‘All in One Well’ Microplate System

Abstract

The purification is based on a set of solutions and a simple centrifugation procedure. Protocols are designed for the easy extraction and purification of genomic DNA from blood samples and tissue cells, including whole blood, buffy coat, bone marrow, body fluids, buccal cells, tissues, mouse tails, etc. Red blood cells are lysed by dilution into a hypotonic solution. Tissues are broken down and treated with protease K to release cells. An anion detergent solution is used for further cell lysis and DNA extraction. After precipitation of the detergent and proteins, a unique bead that bind proteins, lipids, and RNAs are added to achieve the supreme purity. Genomic DNA is then separated by alcohol precipitation. A proprietary nucleic acid precipitation reagent is used to enhance DNA recovery from low cell number samples. No DNA binding beads or column is used in the method, eliminated the problem of low yield and the risk of shearing genomic DNA. The purified samples are free of proteins, lipids, salts, and RNAs contamination, are stable for storage and suitable for all downstream applications.

J Biomol Tech. 2009 February; 20(1): 34.

V9-S1 Multiplex Sequencing on the SOLiD™ Platform

Abstract

The SOLiD™ DNA sequencing system utilizes stepwise ligation of oligonucleotide probes and enables high fidelity, high throughput sequencing. Increases in throughput since release of the SOLiD™ system have been dramatic, yielding more DNA sequence in a single instrument run than is required for certain applications. In order to better match experimental needs to sequencing capacity, we have developed and commercialized a multiplexing method for the SOLiD™ system that enables pooling of 16 unique samples per sequencing spot, or up to 256 samples per two-slide run. Multiplexing of 96 samples has been demonstrated, indicating that the method is scalable to even higher throughput. Multiplexing is achieved by making sequence fragment libraries using a set of oligonucleotide adapters that contain short unique sequences, or barcodes, which are sequenced as a short (5–10 base) read separate from the sample sequence. The barcode sequences are analyzed to parse the sample sequences by their library of origin. Parameters used to design effective barcode sequences include uniqueness, degrees of separation from other barcodes, color balance during SOLiD™ sequencing, and optimization with respect to observed performance characteristics of the platform. Data is presented from experiments using up to 96 barcodes. False assignment rates below 0.01% have been obtained, with over 96% of matching sequenced tags containing a barcode. Applications suited to SOliD™ multiplexing include, but are not limited to, small RNA expression analysis (for which a multiplexing kit has been commercially released), targeted resequencing, bacterial resequencing, and whole transcriptome gene expression.

J Biomol Tech. 2009 February; 20(1): 34.

V10-S1 Sample Quality Diagnosis in a Commercial Resource Company: An Effective Strategy for Sample Assessment

Abstract

DNA Analysis, LLC is a new company located in Cincinnati, Ohio. We provide services related to automated DNA sequencing, fragment analysis and DNA fingerprinting to the academic and corporate research community. A primary goal of ours is to provide quality services while maintaining a close working relationship with our clients. Our company operates more as a commercial resource facility, not as a large high-throughput company. This requires providing customer services rarely found in commercial laboratories. Samples can be analyzed prior to or after processing for sequencing or genotyping. Potential problems that can interfere with achieving a quality result can be diagnosed. We have incorporated a testing strategy that includes: scanning to determine sample concentration and to identify contaminants using the Nanodrop 1000 spectrophotometer, testing for nicked DNA or DNA contamination using the Lonza Flashgel system, and sequencing with universal primers designed to sequence any plasmid template with the ampicillin or kanamycin resistance genes to help assess primer-template related problems. In combination, these testing methods provide information about sample submissions that are communicated to the clients, and are applied in determining a troubleshooting chemistry or procedure. This poster outlines our general process, from the time a researcher submits a sample, to diagnosing sample quality, to relaying the results and relative information to the customer.

J Biomol Tech. 2009 February; 20(1): 35.

V11-S1 SOLiD System Sample Preparation Amenable to High-Throughput Sample Multiplexing.

Abstract

The sequencing throughput of the next generation sequencing platforms can be significantly increased by sample multiplexing, for example by using barcoding. The SOLiD System provides up to 256 barcodes to analyze as many samples in a single run. This new multiplexing technology is expected to dramatically reduce both labor and reagent costs for users. However, barcoding requires parallel processing of numerous samples during library construction until they receive distinct barcodes. The implementation of the conventional sample preparation workflow for treating tens or hundreds of samples at a time would be impractical due to the prohibitive increase of preparation time and labor. This would greatly limit the applicability and acceptance of barcode multiplexing. Moreover, not all of 14–15 major steps of SOLiD fragment library preparation are amenable to automation. Additionally, there are no high-throughput DNA fragmentation instruments on the market which can shear in parallel multiple samples to the size-range required by SOLiD. Here we present a high-throughput sample preparation solution for the SOLiD System. This includes a new DNA fragmentation instrument and a simplified five-step library construction workflow. The new low-volume parallel DNA shearing protocol generates DNA fragments with such a tight size distribution to render size-selection unnecessary. Furthermore, the enzymatic steps of the new library preparation are directly coupled with the DNA shearing process, feature a simple one-tube workflow, and make possible the omission of the large-scale PCR step. Compared to the conventional method, the new sample preparation process is much shorter (less than 3 hours) and results in a greater library yield and quality. It also eliminates any base compositional bias resulting from PCR and over-shearing of DNA, and improves the coverage of otherwise “difficult-to-cover” regions. This method therefore enables sample multiplexing with the SOLiD System by providing the solution for automated sample preparation.

J Biomol Tech. 2009 February; 20(1): 35.

SR4-S1 Human Genome Sequencing Using the AB/SOLiD Platform

Abstract

NextGen platforms are now becoming widely used for human genome sequencing to characterize whole-genome polymorphism events and for functional mutation discovery. As the cost of producing whole genome data approaches $10K/Genome, thousands of human samples could be sequenced, requiring methods for quick data analysis and contamination screening such as eGenotyping. As a participant in the 1000 Genomes and TCGA (The Cancer Genome Atlas) projects we have developed a high throughput pipeline using the AB/SOLiD platform for whole genome sequencing. At the BCM-HGSC, over 320 GB of mapped data have been completed on the AB/SOLiD for whole genome sequencing activities in both mate pair and fragment libraries. Current runs have achieved up to 16GB generated per mate pair run with 58–60% matching reads. New procedures and methods were developed for the SOLiD pipeline to ensure library complexity (90+% unique reads), bead load density (100–120 K/per panel), run transfers, off line analysis and submissions at the sequence generation level. The AB software tool Corona_lite has been used to determine SNPs and small indels for SOLiD data. Initial data on SOLiD 25X coverage data has shown that 100% of HapMap SNPs are found at 12–15X sequence coverage. Building on these pipeline advancements we are now determining the appropriate depth of coverage and analysis needed for whole genome sequencing to identify SNPs and structural variants for human disease related samples including patients with Ataxia, Pancreatic Cancer and Glioblastoma.

J Biomol Tech. 2009 February; 20(1): 35.

SR5-S1 Simplifying Illumina GA Sequencing and Multiplexing Samples as a Result

Abstract

We have developed a simplification to the multi-primer hyb protocols for sequencing DNA and smallRNA libraries on the same Illumina GA flow cell. This simplification requires minimal modifications to current protocols but also allows a degree of sample multiplexing with no change to library prep protocols. A doubling of samples throughput is possible with this approach.

J Biomol Tech. 2009 February; 20(1): 36.

V12-S1 Deep Sequencing-Based Whole Transcriptome Analysis of a Single Cell

Abstract

The next generation sequencing technology is a new powerful tool for transcriptome analysis. However, under certain conditions, only small amount of material is available for analysis, which requires more sensitive techniques that can preferably work on a single cell. Here we developed a digital gene expression profiling assay at single cell resolution by combining a modified single cell whole transcriptome amplification method with the next generation sequencing technique, SOLiD™ System. Using this assay, we showed that blastomeres in a four-cell stage embryo have similar gene expression, which is compatible with the fact that they have similar developmental potential. We proved that, compared to cDNA microarray technique, our single cell cDNA deep sequencing assay can detect expression of thousands of more genes. Moreover, for the genes detected by both microarray and deep sequencing, our assay detect new transcript variants for a large proportion of them, which unambiguously confirms at single cell resolution that the transcriptome complexity is more than traditionally expected. Finally, by using our assay to Dicer knockout and Ago2 knockout oocytes, we showed that a significant amount of transposons were abnormally upregulated in Dicer/Ago2 knockout mature oocytes compared with wildtype controls. Development of this technique will greatly facilitate discovering new genes and understanding transcriptome complexity when material available is very limited, such as during early embryonic development or for stem cells, which are usually rare cell population in vivo.

J Biomol Tech. 2009 February; 20(1): 36.

V13-S1 Low Cost, off the Shelf, Automation of SOLiD™ System Template Bead Preparation with Freedom EVO® and Te-MagS™ from Tecan®

Abstract

Preparation of the template beads for the SOLiD™ System is a tedious manual process with 230 steps per sample needed to prepare the beads for sequencing after emulsion PCR (ePCR). With the ability to sequence up to 16 samples per slide, this translates to 3680 steps. Full preparation of the beads for sequencing can take 3½ days and 4 weeks of training. We have taken two sections from this process that have the most repetitive wash steps and that do not require centrifugation or a hood and automated them with “low cost,” off the shelf liquid handling equipment, Freedom EVO® and Te-MagS™ from Tecan®, for a quick and straightforward solution for AB customers. Of particular concern to researchers is: (1) template bead loss leading to lost sequence; and (2) incomplete washing leaving residual chemicals that could interfere with subsequent reactions. We will present data that will compare bead loss for manual vs. robot handling. We will also present data that compares P2-enriched bead recovery, Noise/Signal Ratio and Cy3 intensity between manual vs. robotic handling. The purpose of this experiment is to verify the consistent performance of the robot over time, and show that no difference is observed between the robot and manual processes.

J Biomol Tech. 2009 February; 20(1): 36.

V14-S1 Modified cDNA Library Preparation Method for Transcriptome Profiling by 454 Sequencing

Abstract

454 Sequencing is well suited for transcriptome profiling because readlengths in the 400–600 bp range can contribute significantly to the discovery and characterization of new splice variants in previously uncharacterized transcriptomes. Many standard cDNA library preparation methods have been used for 454 Sequencing applications with mixed success. Most notably, the classic approach of making oligo d(T) primed full length cDNA, fragmentation by nebulization, followed by ligation to standard 454 shotgun library adapters does not always yield optimal results. This is due primarily to the poor fragmentation of <1 kb cDNA fragments and the potential problems caused by excessive poly A/T signal. We have adapted the existing cDNA library preparation protocol recently described by Noonan et al. for use with 454 Sequencing. The protocol can be used with as low as 200 ng of mRNA input and does not require any adapter ligation. We have used the method to sequence the transcriptome of Saccharomyces cerevisiae using standard GS FLX conditions. Two standard FLX runs generated 193 MB of data. Reads were assembled and mapped to >94% of all predicted ORFs and 58% of ORFs had >90% coverage. Positional coverage of ORFs is slightly lower in 5′ and 3′ as compared to the middle. Coverage of the 5′ and 3′ ends appears to be equivalent. We believe this method will offer an additional option to users of 454 Sequencing interested in transcription profiling.

J Biomol Tech. 2009 February; 20(1): 36–37.

V15-S1 Multiplex Sequencing on the SOLiD™ Platform

Abstract

The SOLiD™ DNA sequencing system utilizes stepwise ligation of oligonucleotide probes and enables high fidelity, high throughput sequencing. Increases in throughput since release of the SOLiD™ system have been dramatic, yielding more DNA sequence in a single instrument run than is required for certain applications. In order to better match experimental needs to sequencing capacity, we have developed and commercialized a multiplexing method for the SOLiD™ system that enables pooling of 16 unique samples per sequencing spot, or up to 256 samples per two-slide run. Multiplexing of 96 samples has been demonstrated, indicating that the method is scalable to even higher throughput. Multiplexing is achieved by making sequence fragment libraries using a set of oligonucleotide adapters that contain short unique sequences, or barcodes, which are sequenced as a short (5–10 base) read separate from the sample sequence. The barcode sequences are analyzed to parse the sample sequences by their library of origin. Parameters used to design effective barcode sequences include uniqueness, degrees of separation from other barcodes, color balance during SOLiD™ sequencing, and optimization with respect to observed performance characteristics of the platform. Data is presented from experiments using up to 96 barcodes. False assignment rates below 0.01% have been obtained, with over 96% of matching sequenced tags containing a barcode. Applications suited to SOliD™ multiplexing include, but are not limited to, small RNA expression analysis (for which a multiplexing kit has been commercially released), targeted resequencing, bacterial resequencing, and whole transcriptome gene expression.

J Biomol Tech. 2009 February; 20(1): 37.

V16-S1 Polymorphism Discovery in High-throughput Resequenced Microarray-enriched Human Genomic Loci

Abstract

Identifying genetic variants and mutations that underlie human diseases requires development of robust, cost-effective tools for routine resequencing of the regions of interest in the human genome. Here we demonstrate that coupling Applied Biosystems SOLiDTM System sequencing platform with microarray capture of targeted regions provides an efficient and robust method for polymorphism discovery in human. Utilizing high-density Agilent microarrays with a custom probe design to pull down 4.3 Mb target DNA sequence from a HapMap Yoruba sample, we obtained sequencing coverage averaging 138-fold, with median coverage equal 59. This level of coverage enabled highly accurate and sensitive SNP detection, with 99.5% of identified HapMap SNPs called correctly. In addition, the enrichment/resequencing strategy allowed precise localization of insertion/deletion and inversion polymorphisms, as well as detailed characterization of fusion breakpoints in chromosomal translocations, in fragment libraries. These results demonstrate that the combination of SOLiD resequencing with microarray capture of the selected genomic regions provides a powerful tool for genetic analysis and will expedite the search for genes contributing to inherited common diseases and the diseases in which somatic mutations play a role, such as atherosclerosis and cancer.

J Biomol Tech. 2009 February; 20(1): 37.

V17-S1 Improving the Quality of DNA Libraries in the Solexa Sequencing Workflow by Implementing a High Sensitivity DNA Quantification Method

Abstract

Next-generation sequencing technologies play an important role in investigating complete cancer genomes and transcriptomes. To further increase productivity of this compelling technique, the quality of DNA libraries plays an important role. One important step in the Solexa workflow is the amplification of the generated libraries for determining the exact sequencing cluster concentration. The drawback of this step is that amplification artefacts and errors will be introduced into the target sequence. An on-chip electrophoresis instrument has become a standard tool for implementing DNA library quality control and quantification in the Solexa workflow. The microfluidic device monitors the size and quantification of the amplified libraries and also helps to detect contaminating artefacts. With an optimized protocol and newly developed electrophoresis chemistry, the sensitivity could be increased by a factor of 20–30, down to the pg/μL concentration range. This improved detection sensitivity allows for the significant reduction of amplification cycles thereby reducing the target sequence error rate.

J Biomol Tech. 2009 February; 20(1): 37.

V18-S1 The Utility of Capillary Electrophoresis and Next Generation Sequencing Platforms for Scientific Discovery

Abstract

With its long read lengths and high accuracy, capillary electrophoresis-based sequencing is the gold standard technology for de novo projects. Typically in de novo projects for genetic analysis of any organism CE is considered ideal for creating high quality scaffolds. Now with the availability of sequencing by short-read next generation sequencing technologies system, this process is complemented through finishing with high coverage. Alternatively, short-read sequencing technologies offer the throughput requirement for assaying large numbers of candidate regions or when resequencing pooled or heterogeneous samples. Next-generation sequencing is suited for large-scale discovery experiments, while CE with its high accuracy and unmatched data quality can be used to validate structural genetic variations. We will to look at de novo and targeted medical sequencing as two applications where each technology could be applied. In our analysis we consider sample preparation, and reagents, as well as key criteria that researchers consistently demand, including accuracy, coverage, read length, quality values and ease of use.

J Biomol Tech. 2009 February; 20(1): 38.

V19-S1 Next Generation Sequencing of the Escherichia coli O55:H7 Genome and Comparison with the Closely Related Enterohemorrhagic E. coli O157:H7

Abstract

Detection in the food supply of pathogenic E. coli, particularly strains that cause hemorrhagic colitis (HC), has become a public health priority. The O157:H7 serotype of E. coli has been responsible for most HC outbreaks to date, so detection of this type is critically important. The ideal assay must detect O157:H7, but not any other serotypes, including the vast majority of commensal E. coli that are not pathogenic. E. coli O157:H7 is very closely related to the O55:H7 serotype, which does not frequently cause HC outbreaks. Numerous lines of evidence indicate that O55:H7 is the nearest phylogenetic neighbor of O157:H7, making the design of O157:H7-specific assays challenging. The E. coli O55:H7 genome sequence would be a valuable tool for identification of assay target sequences unique to O157:H7, but no such sequence was available. To this end, the genome of one E. Coli O55:H7 strain, and another O157:H7 strain, were sequenced by oligonucleotide ligation and detection using the next-generation AB SOLiD™ platform. Comparison of the O55:H7 and O157:H7 genomes identified 500 kb of sequence that is present on the O157:H7 chromosome and absent or divergent in O55:H7. Comparison of these putative O157:H7-specific sequences against the publicly available genome sequences of other pathogenic and non-pathogenic E. coli and Shigella strains identified regions that are conserved beyond the O157:H7 lineage, further narrowing the list of putative assay design targets. The short time requirement (two to three weeks from library construction to sequence) and deep coverage obtained (>60X), makes the SOLiD™ system ideally suited for microbial genome sequencing when a closely related reference genome sequence is available. In particular, this method can be sufficiently robust to permit genome sequencing of a reference organism’s nearest phylogenetic neighbors. Importantly, short-read mapping methods can define regions of difference between the query and reference genomic sequences, which is fundamental to the definition of specific target sequences for differential assay design.

J Biomol Tech. 2009 February; 20(1): 38.

V20-S1 Automation of the GS FLX Titanium Shotgun Library Preparation and Implementation at the 454 Sequencing Center

Abstract

Preparation of a single stranded DNA library is the initial step of the 454 Sequencing™ sample preparation process for many applications. Current manual batch size in the 454 Sequencing Center is 8 libraries/FTE/8 hour day. We have increased production capacity 12-fold by automating the GS FLX Titanium shotgun DNA library protocol to allow the parallel processing of up to 96 libraries/FTE/8 hour day using a commercially available liquid handler. Nebulization has been replaced by fragmentation on a Covaris™ E210 instrument. This allows for the unattended fragmentation of up to 96 samples in 3.5 hours (including set up and break down times). Because there is no DNA loss during fragmentation with the Covaris™ instrument we have been able to reduce the DNA input requirement from 5 to 3 μg of double stranded DNA. All post-fragmentation steps are carried out in a 96-well plate format on a Hamilton MICROLAB® STAR liquid handler. All Qiagen® Min-Elute® purification steps have been eliminated and replaced by purification using a combination of Agencourt AMPure® SPRI® beads and Qiagen® QIAquick® 96-well plate. The processing time from sizing SPRI® to single stranded library takes approximately 5 hours. The automated processing is robust and reproducible. We find library quality to be equivalent to the manual method. The method is currently in production at the 454 Sequencing Center.

J Biomol Tech. 2009 February; 20(1): 38–39.

V21-S1 De novo Assembly of SOLiD™ Colorspace Reads into Large Genomic Scaffolds Using a Velvet-Based Pipeline

Abstract

Next-generation sequencing technologies, such as the SOLiD™ system, provide the ability to sequence entire genomes quickly and cheaply. Although these platforms are well-suited for genome resequencing, many research projects seek to analyze previously uncharacterized genomes for which a reference sequence is not available. The feasibility of genome assembly from short reads has recently been demonstrated, but to date, no tool has been available for the assembly of colorspace reads from the SOLiD™ platform, which offers the highest throughput of any commercially available sequencing platform. To accomplish this task, the Velvet de novo assembly tool* was adapted to handle SOLiD™ colorspace data. Specifically, all reverse complement operations in the Velvet source code were changed to reverse (not complement) in order to accommodate the properties of colorspace reads. In addition, an analysis pipeline was developed to facilitate the preparation of colorspace reads for input to Velvet, and convert the resulting contigs into nucleotide sequences. The latter step is accomplished by a colorspace-to-nucleotide adaptor that corrects colorspace contigs by aligning their component reads, and maximizing concordance of first base calls with the colorspace translation. Using this pipeline to assemble 50-mers from an E. coli DH10B mate-paired library with 147X coverage, we obtained a contig N50 of 2.2 kb and a scaffold N50 of 68.2 kb. This assembly covered 95% of the 4.6 Mb E. coli DH10 chromosome with high accuracy. Notably, the adaptor rescued over 78 kb of sequence that failed to align in colorspace. This computational pipeline, available at the SOLiD Software Development Community (http://solidsoftwaretools.com), provides researchers with an end-to-end solution for de novo assembly of previously uncharacterized genomes. These tools may also be useful for the reconstruction of insertions in resequencing projects, and clustering of expressed sequence tags.

J Biomol Tech. 2009 February; 20(1): 39.

V22-S1 Expression Profiles of small RNAs from Various Tissues Generated by SOLiD™ Sequencing

Abstract

The combination of the SOLiD™ Small RNA Expression Kit (SREK) with the SOLiD Sequencing System presents a unique opportunity to study miRNA expression in a way not previously possible. To demonstrate the power of this approach, we barcoded and sequenced small RNA libraries from ten different human tissues to saturating levels of detection; generating up to 200 million total mappable tags of data. Comparing both independent sequencing runs and libraries indicates the system is highly reproducible and capable of up to 6 logs of dynamic range of detection. To analyze the quantitative ability of both the library method and the sequencing platform we compared tag count data to real-time PCR assays generated using Taq-Man miRNA low density arrays. Fold-change comparisons between platforms show Pearson correlation values of up to 0.95, indicating the system is a valid and accurate profiling tool. Detailed analysis indicates a far greater repertoire of miRNA variants, or isomers, than previously observed suggesting a much broader range of mRNA targets for miRNA-mediated regulation. We have also developed a Support Vector Machine to predict novel miRNAs contained within the SOLiD datasets. Using this approach we have identified hundreds of potentially novel sequence tags. We chose a subset of these novel transcripts and designed custom TaqMan miRNA assays to validate them by real-time PCR analysis. We are able to demonstrate both the presence and expression profile of >50% of the novel sequences, most of which are present at relatively low levels in the ten tissues. Interestingly, we failed to detect nearly all of these novel targets using conventional northern blotting highlighting the need for qPCR sensitivity for validation purposes. This human miRNA expression atlas provides a unique opportunity to understand the sequence complexity and identity of small noncoding RNAs present in a variety of human tissues.

J Biomol Tech. 2009 February; 20(1): 39.

V23-S1 Broad Genomics Services Offered by Complementary Next-Generation Sequencing Platforms

Abstract

The success of commercial sequencing is largely dependent on the capability of a service company to provide broad and cost effective solutions to meet the various needs of its customers. For providers of Next-Generation (Next-Gen) sequencing services, it’s critical to evaluate and practice the synergy created by the combination of different platforms in order to make genomics projects of any size and scope accessible to researchers and hereby, increase the pace of genomics research. At SeqWright, two complementary Next-Gen platforms, the GS FLX system from Roche and the SOLiD system from ABI, were chosen to provide cost-effective genomic solutions for virtually any genomics project. The GS FLX system produces ten-fold longer sequencing reads than other next generation platforms, making this system ideal for de novo genome sequencing as well as targeted resequencing projects. The latest upgrade to GS FLX, known as Titanium, generates more than one million reads and up to 600 megabases of sequence per run in a matter of several hours. For projects requiring larger data sets, the SOLiD system is the system of choice. A full SOLiD run can currently generate more than 100 million reads and nearly 5 gigabases of sequence, and the newest upgrade is purported to produce over 40 gigabases per run. The experience at SeqWright and data generated demonstrate that the dual-system approach allows service provider to offer a variety of genomics services for a broad array of projects, including whole-genome sequencing/resequencing, SNP detection, transcriptome sequencing, non-coding RNA discovery, and ChIP sequencing, etc.

J Biomol Tech. 2009 February; 20(1): 39–40.

SR6-S1 Evaluation of Two Whole Genome Amplification Strategies for the Application of Buccal Cell DNA to High-Density SNP Array Analysis

Abstract

Mouthwash (MW) buccal cell collection and DNA purification is an economical and convenient option for gene association studies, though it typically results in lower DNA yields. Whole genome amplification (WGA) will increase the DNA mass of these samples, but reportedly produces allele dropout. We evaluated the concordance of SNP results between two strategies of WGA DNA from eight buccal cell samples and DNA from eight peripheral blood leukocytes (PBL) samples on Illumina Inifinium HumanLinkage-12 Arrays. WGA was performed on MW DNA per manufacturer’s directions using the QIAgen Repli-g Midi kit (1x) and in four half volume WGA reactions pooled after amplification (4xP). Of the 6090 SNP’s on the array, 71 did not yield results for any sample and were excluded from further analysis. For the remaining 6019 SNPs, call rates were 99.77% for PBL DNA, 90.44% for 1x WGA, and 92.03% for 4xP WGA. PBL DNA heterozygotes were called homozygotes (allele dropout) in 0.12% of both the 1x and 4xP WGAs. PBL DNA homozygotes were called heterozygotes in 0.04% 1x WGAs and 0.03% 4xP WGAs. The kappa value for overall concordance was 0.9975. Using the 36,144 SNP’s with 100% call rates across all samples, allele dropout rates were 0.10% and 0.07% for 1X and 4xP WGAs, respectively. Homozygote to heterozygote miscalls were 0.03% for both WGA strategies. Kappa values increased to 0.9985 for 1x WGAs and to 0.9989 for 4xP WGAs. Overall SNP result concordance between high quality peripheral blood leukocyte DNA and whole genome amplified material from buccal cell DNA was excellent (>99.7%). The 4xP strategy showed some improvements in call rate and accuracy. Despite a reduction in overall SNP success with WGA buccal cell DNA, the high concordance suggests SNPs called should be expected to agree with calls on PBL DNA.

J Biomol Tech. 2009 February; 20(1): 40.

SR7-S1 Advances in SCODA Electrophoretic Biomolecule Concentration

Abstract

Continued development of SCODA, an electrophoretic biomolecule concentration method developed at the University of British Columbia, has led to substantial performance improvements which will be presented. This technology has been commercialized through Boreal Genomics who have developed an instrument for nucleic acid and protein purification with greatly improved performance over early proof-of-concept demonstrations. The SCODA technology offers unique advantages including exceptional rejection of PCR inhibitors and other contaminants, an unparalleled ability to enrich for low abundance nucleic acids, and ability to length-select nucleic acid fragments. Recent advances have reduced sample processing time from ~1 h to under 10 minutes, for samples up to 1 mL in volume. Nucleic acid purification from samples ranging from Athabasca tar sands, various soils and other environmental and food matrices have been demonstrated including high molecular weight DNA recovery when desired. The instrument’s ability to reject contaminants and proteins has been benchmarked against competing column technologies, and has been demonstrated to be 100- to 1000-fold more effective at protein and contaminant rejection, while maintaining high DNA recovery efficiency even in highly contaminated samples. We have also demonstrated the ability to set up instrument conditions such that proteins will focus instead of being rejected, opening the way to a number of protein purification applications. The new alpha instrument will be presented, as will its performance specifications and recent advances in sequence and length specific DNA concentration.

J Biomol Tech. 2009 February; 20(1): 40.

V24-S1 Purification of Genomic DNA with Minimal Contamination of Proteins

Abstract

The purification is based on a set of solutions and a simple centrifugation procedure. Protocols are designed for the easy extraction and purification of genomic DNA from blood samples and tissue cells, including whole blood, buffy coat, bone marrow, body fluids, buccal cells, tissues, mouse tails, etc. Red blood cells are lysed by dilution into a hypotonic solution. Tissues are broken down and treated with protease K to release cells. An anion detergent solution is used for further cell lysis and DNA extraction. After precipitation of the detergent and proteins, a unique bead that bind proteins, lipids, and RNAs are added to achieve the supreme purity. Genomic DNA is then separated by alcohol precipitation. A proprietary nucleic acid precipitation reagent is used to enhance DNA recovery from low cell number samples. No DNA binding beads or column is used in the method, eliminated the problem of low yield and the risk of shearing genomic DNA. The purified samples are free of proteins, lipids, salts, and RNAs contamination, are stable for storage and suitable for all downstream applications.

J Biomol Tech. 2009 February; 20(1): 40–41.

V25-S1 TaqMan® Express Plates: A pre-plated format for easy mRNA quantification

Abstract

TaqMan® Express Plates contain up to 96 pre-spotted and dried TaqMan Gene Expression Assays on an optical 96-well plate. Pre-plated assays simplify, accelerate and help error-proof mRNA quantification experiments. This is especially important when there is more than one operator in a lab, when a study is being done across multiple labs, or when a study is extended over a period of time. The assays in TaqMan® Express Plates are user-selected from a catalogue of >50,000 TaqMan assays targeting human, mouse, rat, Rhesus and dog genes. Here, we show that TaqMan® assays which are aliquoted and dried perform comparably to standard wet assays aliquoted immediately before use (dynamic range, linearity, and limit of detection). The difference between the normalized Ct values of wet vs. dry assays was <0.3. The data also showed strong reproducibility for replicates within and across plates and across manufacturing lots. In addition, excellent reproducibility was observed for plates run by different operators across different laboratories. Finally, we have generated a set of 32 human genes with low expression variability among biological samples. The set includes genes commonly used as endogenous controls in relative quantification experiments, plus genes found in a transcriptome-wide screen of multiple human tissues. The performance of assays targeting these genes was tested using total RNA from 20 different tissues. Analysis of expression stability using the geNorm and NormFinder algorithms shows the newly identified genes have expression stabilities equivalent to, and in some cases exceeding, genes historically used for endogenous controls. The full set of 32 assays is available on the TaqMan® Express Human Endogenous Control Plate, enabling rapid experimental identification of optimal control assays for individual user samples.

J Biomol Tech. 2009 February; 20(1): 41.

V26-S1 WikiLIMS as a Platform for Next Generation Sequencing and Bioinformatics Analysis

Abstract

WikiLIMS is customizable software based on MediaWiki and Semantic MediaWiki that captures data from Next Generation sequencers. It is being used at a number of laboratories to facilitate lab workflows and display data. WikiLIMS users can use WikiLIMS to integrate expression data and sample data with their sequence studies. This poster describes WikiLIMS extensions that read data from caGRID using its WSRF-based services and Perl, and demonstrates that the WikiLIMS extensions can compare sequence data from caBIG with data from the Next Generation sequencer using an SGE computing cluster. WikiL-IMS is also being used as a research desktop, an application to launch a variety of command-line applications, retrieve their output, and create useful, shared bioinformatic work-spaces. This work shows the utility of MediaWiki and WikiLIMS within the framework of publicly available biomedical data.

J Biomol Tech. 2009 February; 20(1): 41.

V27-S1 Next Gen Laboratory Software Systems for Core Facilities

Abstract

Throughout the past year, as next generation sequencing (NGS) technologies have emerged in the marketplace, their promise of what can be done with massive amounts of sequence data has been tempered with the reality that performing experiments and working with the data is extremely challenging. As core labs contemplate acquiring NGS technologies, they must consider how the new technologies will affect their current and future operations. The old model of collecting and delivering data is likely to change to one where the core lab becomes an active participant in advising and helping clients set up experiments and analyze the data. However, while many labs want to utilize NGS, few have the Information Technology (IT) infrastructures and procedures in place to successfully make use of these systems. In the case of gene expression, NGS technologies are being evaluated as complementary or replacement technologies for microarrays. Assays like RNA-Seq and tag profiling that focus on measuring relative gene expression require that researchers and core labs must puzzle through a diverse collection of early version algorithms that are combined into complicated workflows with many steps producing complicated file formats. Command line tools such as MAQ, SOAP, MapReads, and BWA, have specialized requirements for formatted input and output and leave researchers with large data files that still require additional processing and formatting for other analyses. Moreover, once reads are aligned, datasets need to be visualized and further refined for additional comparative analysis. We present solutions to these challenges by showing results from a complete workflow system that includes data collection, processing, and analysis for RNA-seq suited for the core laboratory.

J Biomol Tech. 2009 February; 20(1): 41–42.

SR8-S1 Application of RIP-Chip to Study the Human Regulatory Code

Abstract

Eukaryotic organisms depend on the actions of RNA-binding proteins (RBPs) for successful post-transcriptional control of gene expression. RBPs provide the link between transcriptional and translational regulation and play essential roles in many regulatory processes including transcription, splicing, export, stability and translation. Previously we developed methods for purifying endogenous RBP-RNA complexes and identifying the associated RNA targets using whole-genome expression array technologies (termed ribonomic profiling or RIP-Chip). This advance enabled the large-scale identification of many mRNA targets of RBPs and provided new insight into the principles governing post-transcriptional gene regulation. Our studies demonstrated that, analogous to transcriptional regulation, groups of functionally related RNAs are coordinately regulated in a combinatorial manner by distinct classes of RBPs targeting related cis-regulatory elements located in the transcripts. In the process of developing this profiling methodology, we have evaluated different array formats. These studies compare the effectiveness of these platforms for RIP-Chip based on their probe design, amplification strategies, signal to noise and overall cost. Also discussed will be the quality control metrics developed specifically for immunoprecipitated mRNA samples and potential pitfalls. In conjunction with the NIH/NHGRI ENCODE project we have adapted ribonomic profiling to tiled-microarray platforms to determine the associations of both coding and non-coding RNAs for several RBPs. By combining RIP-Chip profiling with tiling-arrays to explore the post-transcriptional network, our studies indicate that in addition to targeting predicted mRNAs, many of the non-coding RNAs expressed from the genome also appear to be associated with RBPs in a specific and selective manner that is involved in reading our regulatory code.

J Biomol Tech. 2009 February; 20(1): 42.

SR9-S1 Choosing a Microarray for miRNA Analysis

Abstract

There are several microarrays available for miRNA analysis. We present a comparison of six platforms and highlight some of the issues that users should consider for miRNA analysis. The different platforms have varying input RNA requirements, probe design and protocols. We discuss the choice of a platform for use at CRUK’s Cambridge Research Institute.

J Biomol Tech. 2009 February; 20(1): 42.

SR10-S1 Multiplex Amplification and Microarray-Based Normalization for HT Cloning of Microbial Genes

Abstract

The technology to rapidly clone hundreds or thousands of genes in parallel has been developed and refined over the last several years. The Pathogen Functional Genomics Resource Center (PFGRC) has established a robust high-throughput Gateway® cloning platform and now proposes a multiplex strategy to streamline HT cloning. By miniaturizing primer synthesis and multiplexing PCR and cloning reactions we can reduce cost and labor significantly. PCR primers are synthesized on Agilent custom arrays, cleaved, selectively pooled (1250 ORFs/pool) and used in a multiplex PCR reaction. The diverse PCR product pool is then hybridized to a capture array to normalize relative abundance of each product in the pool. The normalized pool then enters a batch recombination reaction using BP clonase; unique clones are sorted and sequence verified downstream using Sanger and 454 sequencing methods. Preliminary results have confirmed the inherent bias in multiplex PCR and our proof-of-concept experiments explore the possibilities of a highly multiplexed cloning platform. Project funded by the National Institute of Allergy and Infectious Diseases (NIAID) under contract no. N01-AI15447

J Biomol Tech. 2009 February; 20(1): 42.

V28-S1 A Novel Digital Technology for Non-Enzymatic Direct Multiplexed Measurement of Gene Expression

Abstract

We have developed a novel digital technology that can be used for non-enzymatic direct multiplexed measurement for 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 micorarrays, TaqMan® and SYBR® Green Real-Time PCR.

Materials and methods.

nCounter hybridization reactions were performed in triplicate with total RNA samples isolated from mock and polio virus infected human A549 cells. nCounter reactions were set up as follows:

  • 100 ng of total RNA
  • Reporter and capture probes for 509 human mRNAs and controls made to non-human sequences (6 positive, 2 negative)
  • DNA control targets spiked in at 0.1, 0.5, 1, 5, 10, and 50 fM

Hybridizations were carried out for 20 h at 65°C. Excess reporters were then removed by using magnetic bead based purification. The same samples and amount of RNA were also analyzed with Affymetrix® U133Plus2 arrays, using the two-cycle amplification/labeling protocol recommended by the manufacturer. We selected a subset of 14 genes in which the measured log2 fold-change was significant in one platform but not the other for further analysis by TaqMan Real-Time PCR.

In a second experiment, nCounter hybridization reactions were performed in triplicate as described above with total RNA samples isolated from sea urchin embryos collected at seven different development time points. A set of 21 genes were selected for comparison with existing SYBR Green Real-Time PCR data generated in the Davidson Lab.

J Biomol Tech. 2009 February; 20(1): 43.

V29-S1 Development of New 1-mm I.D. Divinyl Benzene-Based Monolithic Reversed-Phase Column and Its Applications

Abstract

Monolith columns offer several advantages over conventional porous columns. They include fast mass transfer, high loading capacity, improved resolution even at elevated flow rates and wide pH stability. These characteristics support versatile performance in a wide range of bio-molecule separations. Earlier, we introduced reversed-phase and ion-exchange phases of ProSwift monoliths in 4.6 × 50 mm format. Ion-exchange phases include weak and strong anion and cation exchange phases. Subsequently, we introduced weak and strong anion exchange phases and a weak cation exchanger in 1 × 50 mm format. 1 mm format provides improved sensitivity and reduced solvent consumption as compared to 4.6-mm I.D. format. Another useful feature is that these 1mm monoliths could be run at flow rates up to 0.3 mL/min and therefore can be used on analytical HPLC instruments with minor plumbing modifications using a nanoflow cell detector. Currently, the development of ProS-wift RP 1 mm reversed-phase columns is in progress. They have been tested in two different lengths. While ProSwift RP 1 × 50 mm is planned for high-throughput fast chromatography separations, 1 × 250 mm is intended for complex protein sample analysis and purification. However, either of these columns could be used for protein chromatography and to combine with mass spectrometry applications. In this poster we present various applications including separation of complex proteins, monoclonal antibody (MAb) and other bio-molecules using both ion-exchange and reverse-phased 1-mm columns. We show fast protein and MAb separations that are done under one minute using RP 1 × 50 mm columns. Relevant results including dynamic capacity, ruggedness, and reproducibility of ProSwift monolithic 1-mm columns are presented.

J Biomol Tech. 2009 February; 20(1): 43.

V30-S1 Highly Reproducible Data from a Microfluidic Separation Platform for Application in Discovery Proteomics

Abstract

A microfluidic column and connector system has been developed for high performance chromatography for protein and peptide separations. The microfluidic system was used in conjunction with an Eksigent NanoLC-Ultra and Thermo Scientific LTQ linear ion trap equipped with a New Objectives Nanospray interface. Separation performance for a proteomic standard mixture was compared against a conventionally packed capillary column. For the analysis of complex proteomic mixtures the variability in the observed MS data and protein identification is a result of a combination of diverse factors. The enormous complexity of many proteomic samples can have thousands of peptides in abundances over many orders of magnitude. Online reverse phase separations have peak capacities in the hundreds, and the finite dynamic range of the mass spectrometer (both in terms of electrospray performance and detector considerations) often makes it difficult to achieve reproducible results. To compound this issue, inter-column reproducibility is difficult to achieve for capillary columns that operate at nanoliter flow rates. Inter-column reproducibility is primarily dependent on the column manufacturing process and the installation of these columns into the analytical system. The process of making connections for these nanoliter-per-minute separations is highly skill dependent, and method robustness very difficult to achieve. Microfluidic devices have inherent appeal as a chromatographic medium as the microchannels are defined lithographically and are highly reproducible. We reported recently on a microconnector which facilitates reproducible microfluidic connections at nanoliter flow rates with a very high degree of consistency. In this poster, we will be reporting on our progress on a new microfluidic platform based on this microconnector for nanoliter separations on micro-chips and demonstrate that robust, high quality separations can enhance the reproducibility of proteomic experiments. High run-to-run reproducibility, and low inter-column variation can be achieved, with retention times within 1% in each case.

J Biomol Tech. 2009 February; 20(1): 43–44.

V31-S1 Method for Heart-Cut Analysis Using 2D RP/RP nanoLC for Proteomic Samples

Abstract

Two-dimensional chromatography is often used to separate peptides from proteomic samples in a biomarker discovery workflow. In order to validate a biomarker, many samples need to be analyzed to prove that the same peptides are reproducibly identified and are changing in a statistically significant manner due to a biological perturbation. Rather than running an entire 2D experiment, which is too time-consuming during validation, a better approach is to elute the targeted peptides in one fraction in a heart-cut manner. A highly reproducible method for performing online two-dimensional chromatography with mass spectrometry was developed. Peptides were separated by RP chromatography at high pH in the first dimension, followed by an orthogonal separation at low pH in the second dimension. An online dilution of the effluent was performed after the first dimension so that no hydrophilic peptides were lost in the second dimension. As peptides eluted from the second dimension, a hybrid quadruple time-of-flight mass spectrometer was used to detect the peptides and their fragments by alternating collision cell energy between a low and elevated energy state. All peptides were fragmented in this method, which takes out the irreproducible nature of typical MS/MS experiments. This fragmentation allowed for the identification of the peptides with a novel database searching algorithm that uses 14 physiochemical properties to score identifications and minimize false positives. Proteins had to be identified in two out of three replicate injections, which narrowed the list to the most confident identifications. Comparing the third fraction of a complete 2D experiment to the targeted run in the heart-cut analysis resulted in over 90% of the same proteins and peptides being identified. Comparisons of measured retention times and peak areas between the two methods will be made.

J Biomol Tech. 2009 February; 20(1): 44.

V32-S1 1 mm I.D. Poly(Styrene-Co-Divinylbenzene) Monolithic Columns for Fast and High Efficiency Protein Separations

Abstract

Due to the unique properties of monolithic stationary phases, monolithic columns have become an attractive alternative for packed columns, especially for the separation of complex protein samples. The porous monolith is covalently anchored to the capillary wall. This attachment increases the robustness of the column. The control that can be exerted over the preparation process facilitates optimization of the porous properties of the monolith, and consequently the chromatographic performance of the entire system. Furthermore, virtually no carryover effects are observed on monolithic stationary phases compared to frequent observation on silica materials, resulting in a more reliable identification and quantification of proteins, especially for the low abundant species.

In this study, the LC performance of a 1-mm I.D. monolithic column is demonstrated for the separation of proteins. The effects of gradient time and volumetric flow rate on peak capacity are demonstrated and loadability of the 1-mm I.D. monolithic column was determined for proteins. Furthermore, high efficiency separations of a complex E. Coli protein mixture are demonstrated. For these separations a volumetric flow rate <100 μL/min was applied to ensure high detection sensitivity and maximum compatibility with ESI interfacing and MALDI spotting. Finally, the application of the 1 mm I.D. monolithic column used as a 2nd dimension RP column is demonstrated after an 1D ion-exchange separation.

J Biomol Tech. 2009 February; 20(1): 44.

V33-S1 Separation of Complex Peptide Samples Using Optimized Column Technology and 1D-LC Tonditions

Abstract

Determination of the proteome and identification of biomarkers is required to monitor dynamic changes in living organisms and predict the onset of an illness. Different approaches are available for the identification of proteins. One method is called shotgun proteomics, in which proteins are digested, the resulting peptides are separated by high-performance liquid chromatography (HPLC), and identification is performed with tandem mass-spectrometric detection. Digestion of proteins may lead to a very large number of peptides. For example, it has been estimated that digestion of a cell lysate may produce up to 500,000 peptides. The separation of highly complex peptide samples is one of the major challenges of analytical chemistry.

The effects of LC conditions (gradient time and temperature) and column length on peak capacity for the separation of tryptic peptides was studied in reversed-phase gradient elution nano LC. To perform the study, 75-μm I.D. columns up to 50 cm in length were packed with 3-μm silica particulate materials and tested. Increasing the gradient time and column temperature helped to increase peak capacity. A maximum peak capacity of 443 was obtained with 50-cm-long columns operating with a 120-min gradient time and a column temperature of 60°C.

J Biomol Tech. 2009 February; 20(1): 44.

V34-S1 Development of an Automated Method for Antibodies Purification and Analysis

Abstract

One of the major problems in biotherapeutics is aggregation of the active pharmaceutical ingredient. These product-related substances can have different efficacy than the main product and may cause serious side effects, e.g., anti-drug-antibody formation. Protein aggregates are mostly the consequence of suboptimal production, purification or handling conditions (e.g., temperature, pH). In the purification of antibodies, a protein affinity separation is generally the first step. Affinity chromatography on protein A or G columns typically yields a purity of more than 95% in a single step. To verify the purification efficiency (or sample purity or antibody quality) a technique such as ion exchange or size-exclusion chromatography is needed. Ion exchange stationary phases provide good selectivity for separation of charge variants of the protein biopharmaceutical. The variations may be very subtle or small and finding the optimal chromatographic conditions requires optimization. This work discusses the development of an automated solution for purification and separation of antibodies using a single Ultimate 3000 HPLC system. In this process the autosampler performs the injection, high-volume fraction collection, and reinjection.

J Biomol Tech. 2009 February; 20(1): 44–45.

V35-S1 Plug and Re-Play: How to Divide a Nano LC Flow Post-Column and Conquer the Information Content of Complex Proteomic Samples

Abstract

Here we present a novel low volume splitting T integrated into a 6-port valve to split an analytical nano LC flow post-column. One portion of the analyte flow is directed towards the mass spectrometer immediately, whereas the other portion takes a defined detour and is directed to the mass spectrometer with a time delay, such that a first and second pass of all analytes towards the MS is generated. This brings about two analysis opportunities based upon a single sample injection, a valuable gain in mass spectrometer duty cycle and sensitivity compared to a multiple injection strategy of precious proteomic samples since the mass spectrometer is a concentration dependant detector. This presentation will demonstrate multiple ways to utilize these two analytical opportunities in order to increase the information obtained from complex proteomic samples (e.g., F9 cell lysate). In a non-data-dependent fashion, we can show an increase in protein identification by 20% using either the same instrument setting for both analyte passes (exploiting random information gains), or using CID fragmentation of +2 charge state analytes during the first pass only and ETD fragmentation of +3 charge state analytes during the RePlay run only (exploiting the increased duty cycle of the MS). Furthermore, an information-dependent approach can utilize the first analyte pass, by providing either an immediate database search with a semi-automatic generation of an exclusion list for the second pass of analytes (30% protein identification increase observed), or providing timed SRM settings for the quantification of the identified peptides during the RePlay run, thus allowing for identification and quantification based on the same initial sample injection. This report employed multiple MS systems and their software (LTQ-XL, HCT and QTrap 4000) since, to date, no single system has the combined hardware/software abilities for all modes described here. Flow splitting of nano LC systems post column enables twice the analytical possibilities from the same injection of a complex proteomic sample.

J Biomol Tech. 2009 February; 20(1): 45.

SR11-S1 Surface Plasmon Resonance (SPR) based Sensing of Transfer across Biological Membranes

Abstract

The cell is critically dependent on molecules, ions and signals being continuously transferred across its membrane. Water and other small non-electrolyte molecules can cross the lipid-bilayer membrane by passive diffusion, whereas transport across the membrane of ions and larger molecules are controlled by specialized membrane-spanning proteins. Here, we present, what to our knowledge is the first time-resolved method for direct monitoring of non-electrolyte transfer across biological membranes. The method is based on resolving the temporal change in refractive index upon a permeation-dependent change in the solute concentration inside liposomes attached to a surface plasmon resonance (SPR) active surface. As illustrated for the biologically important molecules glycerol, urea and hydroxyurea, this method, being biosensor-based, enables screening of multiple permeation events on a very same set of immobilized liposome, and a possibility to study selective alterations in lipid bilayer permeability, through in situ injection of effector molecules. Compared to previously available methods, which are based on averring several indirect measurements of solute-transfer induced size changes of liposomes or cells, this method provides a more sensitive as well as time and sample saving mean for studying permeability of non-electrolytes.

J Biomol Tech. 2009 February; 20(1): 45.

SR12-S1 A Robotic Assay for Antibody/Antigen Characterization with MALDI-TOF Mass Spectrometric Detection

Abstract

The National Cancer Institute (NCI) Clinical Proteomic Technologies for Cancer (CPTC) Initiative is a five-year initiative to build a foundation of technologies, data, reagents, reference materials, analysis systems, and infrastructure needed to systematically advance protein biology for the diagnosis, treatment and prevention of cancer. As part of the CPTC initiative, monoclonal antibodies have been raised to potential protein biomarkers to provide a series of renewable and highly characterized affinity binding reagents to the research community.

Our laboratory has utilized a robotic assay based on functionalized magnetic beads to capture individual monoclonal antibodies (mAbs), followed by reaction of the beads with specific antigens to characterize their binding. Following antigen binding, the beads are treated with an acidic MALDI matrix solution to release the antigens and then analyzed by MALDI-TOF mass spectrometry to confirm antibody/antigen specificity. A robotic method capable of the analysis of 96 different mAbs and their potential antigens was written, along with design and manufacture of a MALDI plate holder compatible with the robot and a typical Applied Biosystems Voyager MALDI plate. MALDI-TOF analysis was automated to allow unattended data acquisition.

Several types of modified magnetic beads were evaluated with regard to binding characteristics, background binding, specificity and cost. Among the beads evaluated were carboxylic acid beads chemically modified with polyclonal rabbit anti-mouse Fc antibodies (RAMFc), protein A modified beads, goat anti-mouse IgG modified, and an Fc specific pan-mouse IgG modified surface. Optimal results were obtained using the pan-mouse IgG beads and minimal loading of the target mAb. Antigen concentrations were in the micromolar range with ample signal-to-noise.

J Biomol Tech. 2009 February; 20(1): 46.

V36-S1 Direct Monitoring of Therapeutic Protein Aggregation by MALDI Mass Spectrometry

Abstract

Therapeutic protein aggregates were separated using Size Exclusion Chromatography (SEC) and fractions collected were submitted to a novel high-mass MALDI (matrix assisted laser-desorption/ionization) mass spectrometry (MS) protocol. In order to analyze the biopharmaceutical aggregates intact via MALDI two novel tools were utilized. Firstly, the fractions were cross-linked (K100 Stabilization Kits, CovalX, Zürich, Switzerland) followed by intact detection using high-mass MALDI mass spectrometry (HM1, CovalX, Zürich, Switzerland). A drug-antibody candidate was analyzed which makes tumor cells more vulnerable to chemotherapy and radiation and slows tumor cell growth. Soluble and aggregated SEC fractions were prepared and submitted to cross-linking/high-mass MALDI showing various aggregation states. Candidates exhibiting multimeric aggregation under pressure and temperature stress were analyzed. Aggregated samples were fractionated using SEC and analyzed as the monomeric form and stressed fraction (showing a dimer (306.7 kDa), a trimer (462.9 kDa) and a tetramer (612.4 kDa)). A pharmaceutical formulation sample containing aggregates was directly analyzed (10 μL containing 4 μM) without SEC purification. In the mass spectrum obtained, a large number of proteins are detected due to the absence of pre-fractionation in addition to the monomer and dimer peaks. In order to evaluate the ability to determine the percent of aggregation, a therapeutic protein that is forming a dimer when concentrated was analyzed. A sample containing a dimer was submitted to SEC. The collected fraction corresponding to the monomer and the dimer were submitted to the crosslinking protocol and directly analyzed by high-mass MALDI. The sample corresponding to the monomer was considered as 0% dimer. The one corresponding to the dimer fraction was considered as 100% dimer. After stabilization, the samples collected were mixed and plotted the percentage of dimer as the function of the area of the monomer and dimer peaks (RM and RD). Finally, known samples containing dimmers where analyzed. The described technique provided a rapid tool of measuring therapeutic pharmaceutical aggregates by MALDI mass spectrometry allowing the semi-quantitative measurement of aggregation. These results showed good agreement with the complementary techniques however required a fraction of the sample volume and can be calculated in a fraction of the time.

J Biomol Tech. 2009 February; 20(1): 46.

V37-S1 Using Ion Mobility to Measure Changes in Protein Structure upon Substrate-Ligand Binding

Abstract

Over the past 10 years interest in macromolecule protein mass spectral analysis has increased due to the ability of the current mass spectrometers and electrospray sources to preserve the non-covalent protein/protein subunit interactions, allowing one to analyse proteins in their native conformation and stoichiometry. By coupling mass spectrometry with ion mobility, not only can the intact quaternary mass of biological macromolecules be accurately mass measured, but also the shape (collisional cross section) of the biological macromolecule inferred. All data presented here were acquired on a Synapt HDMS system (quadrupole orthogonal acceleration time of flight mass spectrometer enabled with T-wave ion mobility, Waters, Manchester, UK). Ion mobility calibration was carried out using a mixture of charge-reduced horse heart myoglobin and bovine cytochrome-C. Theoretical collision cross-sections were calculated using the open source-code MOBCAL. PDB files were downloaded from the RSCB Protein Data Bank. We demonstrate that not only can one monitor the change in mass of protein BCL-XL upon binding of a substrate, but also it’s change in collisional cross-section (gas phase shape). We also demonstrate that the gas-phase collisional cross-section of the protein BCL-XL and BCL-XL bound to two different peptide substrates, are consistent with solution-phase NMR data, and that there is a marked increase in collisional cross-section of the BCL-XL protein upon binding of the peptide substrates. We also demonstrate that in the gas phase, we can clearly differentiate and measure the collisional cross sections of the folded protein and unfolded protein BCL-XL, and upon ligand binding, BCL-XL becomes significantly more structured, which is consistent with solution phase NMR data.

J Biomol Tech. 2009 February; 20(1): 46.

V38-S1 Enhanced In-Situ Identification of Peptides Using High-Efficiency Ion Mobility Coupled with Direct MALDI Mass Spectrometry Imaging

Abstract

Imaging mass spectrometry is an emerging tool in proteomics, lipidomics and metabolomics. Biomolecules are analyzed directly from tissue sections, providing spatial information.

Further development of this technique is the identification of diagnostic biomarkers in situ using on-tissue tryptic digestion of proteins followed by MS/MS analysis of tryptic peptides.

However a limitation of MALDI imaging MS is the complexity of the data collected, which can hinder localization and/or identification. In this case a further dimension of separation is required. High efficiency ion mobility separation (IMS), which is based upon separating ions based on differences in mobility passing through a dense buffer gas under the influence of an electric field. Rat brain and human cerebellum (frozen tissue or formalin-fixed-paraffin embedded, FFPE tissue) are sectioned in 10μm thick sections with a cryo-microtome at −20°C, and digested with trypsin using a micro-spotting system (CHIP, Shimadzu) at 34°C during approximately 1 hour of matrix deposition: The alpha-cyano-4-hydroxycinnamic acid matrix in Methanol/TFA 0.1% (50:50) is deposited by a vibrational spraying system (Imageprep, Bruker Daltonics) to cover the brain tissue. All data were acquired on a MALDI hybrid orthogonal acceleration time-of-flight mass spectrometer, (MALDI Synapt HDMS, Waters Corp.). MALDI imaging data from frozen sections demonstrating separation of endogenous lipids and tryptic peptides with nominally isobaric masses using IMS-MS was acquired.

We will show that distinct images can be produced for each of the separated compounds. IMS/MS/MS experiments of several isobaric species will be presented. In addition in FFPE section samples, where mainly peptides are present, we illustrate the different localization observed for different peptides. Identification of several peptides will be shown where direct tissue IMS/MS/MS experiments were performed and whereby nominally isobaric peptides of different intensities could be visualized after IMS.

J Biomol Tech. 2009 February; 20(1): 47.

V39-S1 A Comparison of Manual and Automated Cell Counting Instrumentation

Abstract

Cell counting is a very routine, necessary task in many cell-based laboratory experiments and tissue culture facilities. In order to standardize starting material and to reduce experimental error and variation, researchers should count cells at the beginning of experiments. The hemocytometer is the most widely used device for determining cell concentrations, requiring consistent criteria and tenacity to obtain accurate and reproducible measurements. Regrettably, because the manual method is so tedious, researchers often skip the critical step of cell counting. To facilitate this necessary step, a number of instruments and methods have become available to researchers to facilitate cell counting in routine cell culture maintenance and experimentation. We examined nine different automated cell counting instruments and compared the ease-of-use, throughput capability, cost, accessories and consumables, and performance of these instruments to each other and to glass and disposable hemocytometers. All instruments were operated according to the manufacturers’ instructions, first using a standardized bead solution, then observing relative cell counting and viability data. Several instruments were easy-to-use but the counting and viability data varied significantly from the standard bead solutions as well as from other instrument results. Through-put capabilities were represented by instruments with rapid analysis time (~30 seconds) to 12-sample, hands-off processes. In terms of cost, one instrument was under $5,000, five were under $20,000, and three were over $40,000, and all required the use of consumable accessories. For any given laboratory or core facility, choosing the right instrument will predominately depend upon ease-of-use, needed through-put, and cost.

J Biomol Tech. 2009 February; 20(1): 47.

SR13-S1 Extending MALDI-QqQ-MS Enzyme Screening Assays to Targets with Small Molecule Substrates

Abstract

Mass spectrometry-based high throughput screening has tremendous future potential as an alternative to current screening methods due to its speed, sensitivity, reproducibility and label-free readout. In addition, this method offers a direct readout of the substrate and product, thus minimizing the potential for false positive or false negative hits. We recently reported that a new generation matrixassisted laser desorption ionization-triple quadrupole mass spectrometer (MALDI-QqQ-MS) is ideally suited for a variety of enzyme assays and screening protocols. This instrument provides comparable speeds (at greater than a sample per second) with superior signal-to-background, better reproducibility and a reagent cost savings of greater than 90% as compared to typical fluorescence-coupled assays. Thus far the MALDI-based readout has been validated for a variety of enzyme classes (kinases, phosphatases, proteases, hydroxylases), however all these targets have peptide substrates that are readily monitored without interference from the MALDI matrix. To further extend the application of the MALDI-QqQ readout to enzymes with small molecule, non-peptide substrates, we evaluated this method for measuring enzyme activity and inhibition of acetylcho-linesterase. Due to matrix interference in measuring these small molecules during the MALDI process, multiple reaction monitoring (MRM), available on the QqQ instruments, was used to generate a selective MS/MS transition and accurately measure both the substrate (acetylcholine) and the product (choline) of acetylcholinesterase. Importantly, accurate dose-dependant inhibition measurements were also demonstrated thus validating the MRM readout for enzymes with small molecule substrates and products. Collectively, these data demonstrate that a MALDI-QqQ-MS based readout platform is amenable for small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs.

J Biomol Tech. 2009 February; 20(1): 48.

SR14-S1 Linear Epitope Mapping by Native Mass Spectrometry

Abstract

The identification of antigenic epitopes is critical to elucidating the molecular basis of immune responses. An understanding of binding interactions between antibodies and target antigens is also important for the optimization of monoclonal antibodies intended as therapeutic agents. Mass spectrometry has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antigen-antibody binding and the technique has seen broad application in this field. In this work, we describe a novel methodology for mapping a linear epitope based on direct mass spectrometric detection of antibody (Fab)-antigen complexes under native conditions. To demonstrate the utility of our methodology, we utilized a model system consisting of a Fab with specificity towards Peptide A. Two approaches, epitope excision and epitope extraction, were used in the method. In epitope excision, the Fab and Peptide A complex was treated with a variety of enzymes including: Lys-C, Glu-C, carboxypeptidase B, trypsin and chemotrypsin, and the digested complexes were fully desalted and directly monitored by mass spectrometry. Mass differences between the Fab-Peptide A complex and Fab revealed the size of epitope peptides that were protected by the binding interactions. Using the epitope extraction approach, Peptide A was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data from both techniques allowed mapping of the epitope to amino acid residues 16–27 in Peptide A which is in good agreement with previously reported results. Combining the method with nanospray MS yielded a detection limit in the sub-picomolar range.

J Biomol Tech. 2009 February; 20(1): 48.

V40-S1 Separation of Peptide Precursors from Background Ions and Species with Different Charges by Automated Ion Mobility and Tandem MS Experiments

Abstract

An established technique for characterizing proteins involves tryptic digestion followed by data dependent LC-MS/MS. Complex biological mixtures exhibit a wide dynamic range and the majority of peptides are in the lowest order of magnitude detectable. Despite tandem MS experiments, singly charged chemical noise in the MS spectrum can hinder precursor identification. Cross-linked peptides containing >2 charges exhibit low stoichiometry compared with tryptic peptides, and as such it may be difficult for the mass spectrometer to identify these in the MS survey as candidate precursors. Ion mobility spectrometry (IMS) separates by drift time and m/z giving increases in signal-to-noise ratio of low level species as they separate from the noise, allowing the mass spectrometer to more clearly identify them as candidates for MSMS.

All data was generated using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer incorporating a travelling wave ion mobility separation stage. Species from protein tryptic digests were introduced to the MS by separation on a nanoscale UPLC system or by direct infusion, both coupled to a nanoelectrospray emitter. The system processes and displays a specific band of the m/z versus drift time plot and species within this region are possible candidates. A further criterion of ion intensity was used to select precursor ions for tandem MS interrogation. These selected precursors were isolated by the quadrupole and, whilst maintaining ion mobility separation, were subjected to CID fragmentation by elevating the collision energy either before or after the IMS device. Examination of the data contained in a m/z versus drift time plot shows that clear separation of tryptic peptides based upon charge state can be achieved using an ion mobility device. By programming the mass spectrometer to only consider the selection of species in the area containing species of charge state 2+ or greater, we have analysed a dilution series of a standard four protein tryptic digest (bovine serum albumin, phosphorylase b, alcohol dehydrogenase, and enolase) to show how the enhanced signal to noise of low level species detected by the mass spectrometer in survey scans can lower the limits of detection of the tandem MS/MS approach. In addition, the subsequent separation of precursor ions from background ions by IMS prior to CID leads to high quality MS/MS data of these species. We will show data from the injection of less than 100 amol on column of the four protein digest mixture—these amounts being at a level where a TOF only survey identification is hindered by the presence of the background ions. In the case of cross-linked peptides, a tryptic digest of Bovine Serum Albumin was prepared without reduction and alkylation. This preserves the disulphide bonds and produces numerous large peptides similar in size and structure to chemically cross linked species. We will show that the disulphide linked peptides are ion mobility separated from the more intense doubly charged species present in the digest. Then, by selecting the region of the m/z versus drift time plot containing species with three or more charges, the mass spectrometer is programmed to generate MSMS spectra specific to the disulphide intact peptides contained within this region. For example, data dependent MSMS spectra have been generated on large peptides at approx m/z 1038 (3+), 1298 (3+) and 1500 (4+).

J Biomol Tech. 2009 February; 20(1): 49.

V41-S1 Minimization of Atmospheric Background Contaminants in Off- and On-Line Nanoelectrospray: Identification and Optimization

Abstract

The high surface area of the (sub)micrometer droplets generated by low flow nanoelectrospray ionization results in the potential ionization of contaminants present in laboratory air (Volkmer-Engert R., Schlosser A. J. Mass Spectrom. 2003; 38: 523–525). Neveu and co-workers recently reported (Proceedings of the 56th ASMS Conference, June 1–5, 2008, Denver, Colorado) an active background ion reduction (ABIRD) “bath gas” system designed to reduce these background levels. Here we report on the implementation of a similar system in combination with both off-line nanospray and on-line Nano-LC/MS. The bath gas system was implemented on a modified digital control nanospray source (PicoView, New Objective, Inc.) mounted to a conventional 3-D ion trap (LCQ Deca, Thermo Scientific, Inc.). Mobile phase (water, acetonitirle gradient, 0.1% formic acid) was delivered by a gradient nanoflow LC (Eksigent). Samples for LC analysis (75-μm PicoFrit column, New Objective) were injected with a high performance autosampler (Nano PAL, Leap Technologies). System effectiveness at the suppression of these background contaminants along with other commonly observed background ions, arising from commonly encountered industrial, laboratory, and personal care products, has been studied. The commonly observed background ions (371, 445 m/z) were positively identified as cyclo-siloxane compounds through the use of reference standards. The reduction of ion current from siloxane contaminants of 10-fold or greater was commonly observed with a highly aqueous mobile phase composition (<10% ACN). Background levels are readily reduced to a level suitable for removal of these ions from the typical data dependent mass exclusion list. A systematic study of operational parameters (gas flow rate, composition, etc.) were determined to minimize background ion current without compromising analyte ionization. Air was found to be superior to nitrogen for use with gradient elution LC since air exhibited a higher breakdown voltage and subsequently less chance of corona discharge at the nanospray emitter.

J Biomol Tech. 2009 February; 20(1): 49.

V42-S1 Image Mapping of NanoESI Ion Current with an Automated Digital Control Positioning System

Abstract

Determination of nanospray emitter position for optimal signal is a trial-and-error process that involves adjustment of both emitter position and ESI voltage. We previously reported a feedback controlled source where voltage or emitter position is under feedback control (Valaskovic, et. al. JASMS, 2004, 15, 1201). Here we investigate a similar digital control system to map the spray current of the plume for differing experimental conditions (flow rate, applied voltage, distance). Total and selected ion currents are generated via the systematic three-dimensional raster scanning of the nanospray emitter relative to the spectrometer inlet. Digital PicoView Acquire software (New Objective, Inc.) was modified with a scanning module generating a step motor driven, scan pattern of the emitter with respect to the spectrometer (LCQ Deca, Thermo Scientific) inlet. Each movement to an emitter (X, Y, Z) position triggered MS data file acquisition. After conversion of RAW-formatted files to mzXML, a parsing and data visualization program (LabView, National Instruments) reconstructed data into a mass filtered ion current image map. Images were typically 2 × 2 mm with a pixel step size of 200 μm. The acquisition of a 100 point (10 × 10) data set was on the order of 30 minutes. Signal from a sample containing a singly-charged low molecular weight drug (Buspirone) and a multiply charged peptide (Angiotensin) in aqueous-organic mobile phase, delivered by continuous infusion (340 nL/min) was acquired. Surprising differences in ion map intensity between the singly charged drug molecule (386 m/z), the triply charged peptide (433 m/z), and the doubly charged (649 m/z) ions were observed. Sampling at the edge of the plume showed a distinct relative increase in the population of the +3 ion. The data suggests that emitter position might be exploited in parameter control to selectively favor higher charged peptide ions.

J Biomol Tech. 2009 February; 20(1): 49–50.

V43-S1 Localization and Identification of Lipids by MALDI Tissue Imaging Using Ultra-High Resolution Mass Spectrometry

Abstract

MALDI tissue imaging is a technique used extensively in the protein biomarker field. However, in the low mass range up to m/z 1000 matrix clusters dominate MALDI spectra rendering specific analysis of small molecules difficult. Ultra-high resolution MALDI-Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometers can overcome that lack of specificity of lower resolution/mass accuracy technologies that is mandatory for small molecule imaging and identification. FT-ICR (resolving power >100,000) enables the identification of compounds directly based on high accuracy molecular weight determination providing a global analysis approach eliminating the need for morerestricted MS/MS analyses. Differentiating tissues by small molecules only a few mDa apart gives rise to tracking lipids directly in the tissue. Tissue slices were covered by MALDI matrix and mass spectra of different tissues were acquired with a 9.4 T FT-ICR mass spectrometer equipped with switcheable ESI/MALDI ion source. MALDI images were acquired in positive and negative ion mode depending on tissue type and analytes of interest The 200 Hz smart-beam laser was focused to 40-μm spot size for image acquisition. The classification of different lipids with regard to headgroup and fatty acid composition was achieved based on high resolution high mass accuracy MS analysis alone. Some obtained lipid structures were additionally confirmed by in situ MS/MS analysis. This approach provides simultaneous high resolution images from a larger number of small molecules in a single dataset.

J Biomol Tech. 2009 February; 20(1): 50.

V45-S1 Automated Optimization of Triple Quadrupole Parameters for Nanoflow LC/MS of Peptides

Abstract

Existing automated method development tools for triple quad MRM parameters optimization are based primarily on direct infusion of samples into a mass spectrometer ion source. For more complex samples and standard mixtures, ion suppression and adduct formation often accompany direct infusion methods and complicate the optimization routine. The optimization may fail for many compounds and results for successful ones are often far from optimum. This greatly limits the applicability and reliability of the results. This work explores an optimization routine designed specifically for peptides that allows for chromatographic separation, thus reducing these limitations and obtaining acquisition parameters very close to the optimum values that would be obtained by manual operation by an experienced user. The chromatography-based MRM parameters optimization was tested on synthetic peptides and on tryptic peptides from digestion of standard proteins. Acquisition parameter optimization focused on collision energy values as ion optic settings are generally the same for peptides. For each peptide tested, three transitions were preferred for each precursor with all transitions generally at a higher m/z than the precursor for optimal selectivity. For the synthetic peptides, 30 LC/MS analyses were done to examine collision energy in one-volt steps. Based on this, an automated routine was developed to optimize the predicted transitions for each peptide without operator intervention. A comparison of the automated and manual obtained optimization values will be presented for the selected peptides.

J Biomol Tech. 2009 February; 20(1): 50.

V47-S1 Specific Detection of Proteins by Immunoprecipitation Combined with High Sensitivity Protein Sizing on Microchips

Abstract

Today, immunoaffinity is a crucial tool for the targeted analysis of proteins in complex samples. Techniques like Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting are widely used for a wide range of applications such as biomarker candidate verification in body fluids or clone selection for recombinant protein expression. Here we present a new method that combines the specificity of an immunoprecipitation approach with the high sensitivity of protein detection on microchips using the High Sensitivity Protein 250 assay for the Agilent 2100 bioanalyzer. Initially, sample proteins are derivatized with a fluorescent dye. After incubation with the specific target antibody, the immunocomplexes are captured with Protein A/G coated magnetic beads, washed and eluted by heat denaturation in the presence of SDS. Samples are then directly loaded on microchips and analyzed automatically with the bio-analyzer for protein size and quantity. The final on-chip analysis takes about 30 min for 10 samples and yields digital data. Together with the sample preparation steps, the total assay time is about 3 hours. Results are compared to Western Blotting and pros and cons of both methods are discussed.

J Biomol Tech. 2009 February; 20(1): 50.

V48-S1 Improved Peptide Identification with an Ultra-High-Resolution Quadrupole Time-of-Flight MS

Abstract

Q-TOFs are widely used working horses for proteomics due to their superior mass accuracy in MS as well as in MSMS, making them ideal instruments for de novo sequencing and PTM characterization. For peptide identification at very low concentrations or for highly complex samples, MSMS sensitivity and duty cycle become especially critical. A new designed ultra-high-resolution Q-TOF MS shows significant improvements for these types of samples. In contrast to small molecule applications where MSMS sensitivity is usually defined via signal to noise of a single fragment, peptide identification has additional requirements: Completeness of a fragmentation pattern is more crucial than optimized intensities for single masses. Thus generation of fragment ions covering a broad mass range and their efficient transfer from the collision cell into the TOF part of the instrument were optimized. Using a higher order multipole as collision cell and an additional ion cooler for guiding ions into the orthogonal acceleration showed a dramatic improvement for proteomics applications. As a model system for samples of high complexity, tryptic digests of E. coli, separated using a 90-min nanoLC gradient were used, leading to 7000–10,000 fragment spectra and more than 600 significant protein identifications from 100-ng sample. For single protein digests, analyzed with nanoLC, high sequence coverages could be obtained from sub fmol amounts.

J Biomol Tech. 2009 February; 20(1): 50–51.

SR15-S1 Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell and Mast Cell Signaling

Abstract

Here we introduce and evaluate a new quantitative approach for phosphoproteomic analysis of signaling pathway structure. Our approach combines genetic analysis of isogenic signaling pathway mutants with a quantitative phosphoproteomic method that examines disruption of downstream phosphorylation events through a time course of receptor activation using recently developed visual pathway analysis tools. This new approach is evaluated in the context of the T cell signaling pathway and a T cell clone lacking the upstream Zap-70 tyrosine kinase and its reconstituted counterpart. In our approach, label free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Metabolic labeling of proteins using the SILAC method allows for the calculation of ratios used to compare Zap-70 null and reconstituted cells across a timecourse of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to the critical T cell signaling protein Zap-70. Quantitative phosphoproteomic signatures indicative of downstream inhibition, downstream activation, and mutant compensation are revealed and validated with proteomic pathway visual analysis tools using the established T cell signaling pathway structure as a scaffold. New predictions of the structure of T cell signaling pathway are provided at the phosphorylation site level. The approach described here seeks to transcend the publication of phosphoproteomic data with minimal biological analysis in favor of a targeted approach that positions the deluge of newly discovered phosphorylation sites relative to canonical signaling landmarks. Our methods are adaptable to any cell culture signaling system in which isogenic wild-type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.

J Biomol Tech. 2009 February; 20(1): 51.

SR16-S1 Microwave-Assisted Dephosphorylation (Beta-Elimination and Michael Addition) for Phosphoserine/Threonine Site Mapping

Abstract

Phosphorylation is a common post-translational modification (PTM), which can direct the activity and functions of proteins. The progression of many oncological pathways are dictated by kinase/phosphatase activity and phosphospecific events, hence phosphorylation mapping is currently one of the most active areas of proteomics research. Here, we expand on an alternative method for precise mapping of phosphorylation sites involving microwave assisted β-elimination and Michael addition. Selective modification of these residues to more stable S-ethylcysteine (phosphoserine) and β-methyl-S-ethylcysteine (phosphothreonine) derivatives is a useful tool for determining the exact location of a phosphorylation site by Edman degradation. The nucleophile of choice was 1-propanethiol due to the fact that it was most compatible with our routine chromatographic separation on an ABI 494 Procise Sequencer. 1-propanethiol (76 Da) shows a net change of −22 Da (−98 Da from neutral loss of phosphoric acid and addition of 76 Da) after successful nucleophile incorporation. Traditionally the nucleophile derivatization reaction is performed in the water bath for 1 to 3 h at 60°C. We have found that equivalent results may be obtained by microwave incubation for 2 min at 100°C for protein samples in liquid or blotted on PVDF membrane. We demonstrate that derivatized protein on PVDF membrane can be subsequently Edman sequenced for mapping of specific phosphorylation sites, offering the investigator yet another option for elucidation of phosphorylation sites either near the N-terminus or internally after limited on-membrane enzymatic digestion.

J Biomol Tech. 2009 February; 20(1): 51.

SR17-S1 TiOx Nanostructured Film MALDI Plate for Capture of Phospho- and Glyco-Peptides

Abstract

Nanostructured TiO2 films were grown by depositing under high vacuum a supersonic seeded beam of TiOx clusters produced by a pulsed microplasma cluster source, providing a porous specific substrate which enables selective capture of phosphorylated and glycosylated peptides. Tryptic digests of a six-protein mixture containing phosphoprotein β-casein and glycoprotein fetuin were used as analytes. Phosphopeptides were found to ionize best with alpha-cyano hydrocinnamic acid as a matrix, while the glycopeptide matrix was 2,5-dihydroxy benzoic acid. Several matrix solvents, wash and surface-elution solutions were tested.

Signal for the phospho- and glycopeptides is increased as a result of steps of: deposition, capture, wash of non-specific peptides—all done in-situ on the TiO2 MALDI coated plate. For β-casein, we observe the singly-charged 2061 Da phosphopeptide. For the fetuin, we observe sialylated peptides in the >3000 Da region. Ideal solution for matrix dissolving is one with lowest pH. Best wash solution for non-specific peptide and contaminant removal is one with highest acetonitrile content. Elution of TiO2 captured peptides by depositiong ammonia solution is not necessary. Film thickness range tested were from 50–200 nm, with 200 nm being the ideal. The TiO2 film was also tested for MALDI analysis of standard peptides used to calibrate the TOF/TOF. Performance was exactly as that of the stainless steel plate, showing that both the calibration and enrichment can be done from the same TiO2 coated plate. Results clearly demonstrate that the TiO2 surface concentrates both phosphopeptides and glycopeptides containing sialic acid from enzymatically digested proteins, along with allowing the same level of MALDI ionization for any peptide needed in calibrating the mass spectrometer. This eliminates need for (a) on-column separation/desalting prior to MALDI analysis (b) a separate MALDI plate for calibration.

J Biomol Tech. 2009 February; 20(1): 52.

SR18-S1 Application of In-gel IEF-LC-MS/MS to Human Phosphoproteomics

Abstract

This presentation will describe application of an in-gel IEF-LC-MS/MS analytical strategy for the characterization of human phosphoproteomes. Large-scale analysis of protein phosphorylation in vivo is a highly challenging undertaking that requires powerful analytical and bio-informatics tools. In our phosphoproteome research, we have recently adopted a new methodology that combines in-gel IEF, immobilized metal ion affinity chromatography (IMAC), and LC-MS/MS. The strategy encompasses seven steps: (1) extraction of proteins from the biological system under study; (2) separation of the protein mixture by IEF in an immobilized pH gradient (IPG) strip; (3) sectioning of the entire IPG strip; (4) digestion of the proteins in each gel section; (5) enrichment of phosphopeptides by IMAC; (6) analysis of the enriched digests by LC-MS/MS; and (7) identification of the phosphopeptides/proteins through database searches, and assignment of the sites of phosphorylation in these proteins. The methodology relies on a proven IEF technology with commercially available IPG strips. Because the information about the apparent pI of the identified phosphoprotein is preserved, data from in-gel IEF-LC-MS/MS can be linked to 2D-PAGE data. In our work, we have applied in-gel IEF-LC-MS/MS to the study of the phosphoproteomes in two biological systems: (1) human pituitary tissue, and (2) LNCaP human prostate cancer cell line. In the analysis of the human pituitary, we have characterized a total of 50 phosphorylation sites in 26 proteins. The findings include discovery of novel phosphorylation sites in important pituitary hormones, including the growth hormone, proopiomelanocortin, and proteins of the secretogranin family. In the study of the LNCaP phosphoproteome, we have characterized over 600 different sites in 296 proteins. The characterized phosphoproteins are functionally diverse and include kinases, co-regulators of nuclear receptors, and proteins relevant to cancer. In summary, in-gel IEF-LC-MS/MS is a powerful analytical platform suitable for large-scale characterization of phosphoproteins in complex systems.

J Biomol Tech. 2009 February; 20(1): 52.

SR19-S1 Using Targeted Proteomics to Assess the Impact of Sirtuins on Liver Metabolism in Obesity

Abstract

The NAD-dependent deacetylase SirT1 regulates lipid and glucose metabolism in liver and increased SirT1 activity in caloric-restricted models has been linked to extended life span in several species. Deacetylation of transcription factors and co-modulators, such as p53, NF-κB, C/EPBβ and PGC-1, allows SirT1 to sense and regulate energy levels. The SirT family can also deacetylate mitochondrial proteins, suggesting that posttranslational modification by sirtuins may have global effects on energy metabolism especially gluconeogenesis and lipogenesis. Livers from mice fed either control or a high fat (HF) diet (45 kcal% as fat, 12 wks) were harvested and homogenized. SirT1 levels were measured in whole cell lysates by Western blot. Proteins modified by lysine acetylation were immunoprecipitated with anti-acetyllysine antibody and subsequently separated by one dimensional gel electrophoresis. Bands showing differential staining between the control and HF fed mice were excised and proteins were digested with trypsin. Tandem mass spectrometry using an Agilent Ultra quadrupole ion trap generated product ion spectra that were searched with SpectrumMill against the SwissProt database. The levels and acetylation of identified proteins were validated by immunoprecipitation and Western blotting. Mice on the HF diet were obese, with fatty livers and reduced SirT1 activity as assessed by the NAD+/NADH ratio. SirT1 protein expression levels did not significantly change, however. Acetylation of a subset of the proteins identified, such as carbamoyl-phosphate synthase, uricase, pyruvate carboxylase and ATP synthase, has been previously reported. Interestingly, peroxiredoxin, catalase, and Hsp70, proteins involved in redox and the stress response, were hyperacetylated in the livers of obese mice. We postulate that modification of those proteins could influence the ability of obese mice to modulate oxidative stress, gluconeogenesis and lipogenesis. Globally surveying lysine acetylated proteins using immunoprecipitation and gel electrophoresis/tandem mass spectrometry provides insights into how obesity impacts liver metabolism.

J Biomol Tech. 2009 February; 20(1): 52–53.

V49-S1 Mapping of Disulfide Bonds in Salmon Egg Lectin 24K Using MALDI MS and MS/MS

Abstract

The carbohydrate-binding properties of lectins and their lack of enzymatic activity make this class of compounds “biological adhesives” of great importance. Lectins are invaluable for cell-cell and cell-matrix interactions. Lectins also play significant roles in the fertilization of eggs in higher animal species. There is mounting evidence that egg lectins provide a block to polyspermy via binding to glycoprotein ligands. Block to polyspermy is vital to ascertain proper procreation of species. Because of its superior sensitivity, mass spectrometric detection of peptides has become one of the most popular analytical methods in proteomics. Mass spectrometry allows not only the detection of peptides in minute quantities, but also the structural analysis of post-translational modifications such as phosphorylation, glycosylation, and oxidative disulfide bond formation. The disulfide bond pattern in the salmon egg lectin (SEL24K) from the Chinook salmon Onchorhynchus tshawytscha is presented. The disulfide bond pattern was established with a multi-enzyme digestion strategy in combination with MALDI-MS and MS/MS mass spectrometry. The disulfide bond pattern was found to be symmetrical in the tandem repeat sequence of SEL24K and is fully consistent with symmetrical bond patterns found in egg lectins from closely related fish species with highly conserved locations of cysteines. All cysteine residues were disulfide linked.

J Biomol Tech. 2009 February; 20(1): 53.

V50-S1 Chip-Based Enrichment and Identification of Phosphopeptides

Abstract

Comprehensive identification of protein phosphorylation is a challenging application in the proteomics field due to the complexity and low abundance of phosphorylation events. Researchers have tried various enrichment approaches to simplify and enrich the phosphopeptides before analyzing with LC/MS/MS. Recent advances in microfluidic technology have provided an opportunity to allow easy automation of phosphopeptide enrichment and subsequent analysis of phosphopeptides. We have developed a re-usable microfluidic chip with an enrichment column consisting of titanium dioxide particles sandwiched between reverse-phase materials for on-line selective phosphopeptide enrichment. This approach allows robust, easy-to-use and reproducible enrichment of phosphopeptides from complex matrices. The chip also gives the user options of analyzing the unbound peptides. Specific enrichment of phosphopeptides from Casein, MAP kinases and complex biological samples will be demonstrated.

J Biomol Tech. 2009 February; 20(1): 53.

V51-S1 N- and O-Deglycosylation of Glycoproteins with Subsequent Proteomic Analysis

Abstract

Glycoproteomics is an expanding field with many challenges. There are several techniques available for deglycosylation to allow the study of the glycan and protein separately. Unfortunately, these methods don’t always result in both glycan and protein that are suitable for analysis. It is possible, using PNGase F, to release and analyze N-linked glycans, then do further proteomic analysis of the protein. However, if one is interested in O-glycosylated proteins and their glycans, the most common O-glycan release technology, alkaline beta-elimination, usually destroys the parent protein rendering further proteomic analysis impossible.

We present a method that allows for the separate release of N- and O-glycans, leaving the parent protein relatively undamaged. The fully deglycosylated protein can then be tryptically digested for peptide analysis by LC-MS/MS. This method is unique in that it leaves the glycans and the protein intact for analysis after beta-elimination. The control proteins fetuin and glycophorin A were deglycosylated with PNGase F to release the N-glycans. After the N-glycans were isolated, the proteins were O-deglycosylated using a proprietary, non-reducing beta-elimination reagent. The O-linked glycans were then separated from the proteins using standard techniques. The N- and O-linked glycans were permethylated using a solid phase permethylation protocol and analyzed by MALDI-MS. The deglycosylated proteins were tryptically digested and the peptides analyzed by LC-MS/MS. Glycans were analyzed and identified as the known structures from the proteins. Sequence coverage of the deglycosylated protein was as good as or better than the analysis performed on the intact glycoprotein, proving preservation of the proteomic value of the sample. This analytical workflow can be reversed, such that the O-glycans may be released first, followed by the N-glycans with subsequent proteomic analysis. We will illustrate the analysis of both the N- and O-glycans as well as the peptides generated from the deglycosylated proteins.

J Biomol Tech. 2009 February; 20(1): 53.

SR21-S2 Qualification of a Sensitive Assay for Determination of Norleucine Using Post-Column Amino Acid Separation and Ninhydrin Detection

Abstract

Norleucine (Nle) misincorporation for methionine, which occurs in E. coli expression systems, requires an assay which is sensitive, robust and specific. De novo biosynthesis of leucine in bacterial expression systems leads to a minor amount of norleucine production, particularly in leucine-depleted fermentations. Since the side chain of Nle is similar enough to methionine (Met), some of the tRNAMet will be acylated by norleucine, leading to Nle incorporation at Met positions. Amino acid analysis is one of the few easily adapted methods which can detect norleucine at trace levels. We have developed an amino acid analysis method which detects norleucine at approximately the 10 pmol level and is capable of quantification at the 30 pmol level. A detailed description of the method qualification including controls, system suitability, accuracy, and precision will be provided.

J Biomol Tech. 2009 February; 20(1): 53.

SR22-S2 Vertebrate Courtship Pheromones: Assembly PCR for Codon-Optimized Expression of a Highly Disulfide-Bonded 7 kDa Protein in Pichia Pastoris

Abstract

Plethodon shermani is a species of salamander native to the mountains of western North Carolina that exhibits a complex courtship ritual known as “Tail Straddling Walk.” The courtship enables the male to deliver non-volatile proteinaceous pheromones to a female’s nares that bind to receptors in the vomeronasal organ, thus increasing her receptivity to mate and reducing courtship time. The pheromone extract is composed of two major components: a 22-kDa family called “Plethodontid Receptivity Factor” related to IL-6 cytokines and a 7-kDa hypervariable family known as “Plethodontid Modulating Factor” (PMF) that contains 4 disulfide bonds and is related to snake venom cytotoxins and other three-finger proteins. Three-finger proteins represent a complex group of highly disulfide-bonded proteins serving a plethora of biological functions; however, all reported attempts at recombinant bacterial expression have resulted in disulfide bond scrambling and dimerization. The aim of this study was to produce and characterize a biologically functional, recombinant form of PMF using the methylotrophic yeast Pichia pastoris. A two-step assembly PCR was performed to produce a codon-optimized PMF gene for Pichia. The gene was cloned into a vector containing the Saccharomyces α-mating type secretion signal and transformed into chemically competent Pichia pastoris cells. Expression was performed in shaking culture at 28°C using 1% methanol for induction. Recombinant proteins were purified by AX- and RP-HPLC and characterized by mass spectroscopy. Of the multiple conformations of rPMF that were produced, one was identical to the native PMF. These inexpensive and straightforward protocols may be appropriate for a variety of disulfide-bonded proteins and peptides.

J Biomol Tech. 2009 February; 20(1): 54.

SR23-S2 Protocol Development for Protein Extraction from Sediment Bound Microbes for Bioremediation Purposes

Abstract

Sediments contaminated with heavy metals pose a significant risk to human health and the environment. Remediating these contaminated sediments has historically been difficult due to the cost and labor involved. Bioremediation offers an approach for cleaning up pollutants by enhancing the same biodegradation processes that occur naturally. For example, certain sediment microbes have the capacity to reduce heavy metals into a non-soluble form which prevents them from leaching into waterways. Characterizing these sediment microbes in terms of protein expression provides information needed to enhance microbial nutrient factors for reducing heavy metals and to better understand the unique biological pathways that make these remediation processes possible. Rifle, Colorado, is home to a Uranium Mill Tailings Remedial Action (UMTRA) site where these processes are being studied. A significant challenge in studying microbe proteins from the UMTRA site is their initial removal from sediment particles. Aspects such as the isoelectric point (pI) of the proteins, surface properties of the highly variable sediment particles, biofilms, electrostatic and hydrophobic interactions, and van der Walls and entropic forces among others all play a major role in achieving effective protein removal for proteomic analysis. We will be presenting results from ongoing investigations of various methods for removing microbes specifically from Rifle sediment with special emphasis on methods that are compatible with tryptic digestion and mass spectrometric analysis. These methods include treatments with amino acids and metal oxides to block binding sites of the protein prior to lysis, salts to weaken cross-linking electrostatic interactions, chelating agents to break cross-linking multivalent cations, surfactants to disrupt hydrophobic interactions, and chaotropic agents to desorb proteins from sediment by breaking hydrogen bonds.

J Biomol Tech. 2009 February; 20(1): 54.

SR24-S2 Analysis of the C-terminal Amino Acid Sequence, Glycosylation Site and Disulfide Bond Pattern of Recombinant ISU302 by MALDI-TOF MS and Edman Sequencing

Abstract

Glucocerebrosidase is an enzyme that catalyze the cleavage of the -glycosidic linkage of glucosylceramide. The deficiency of the enzyme in patients with Gaucher disease results in the accumulation of excessive quantities of glucosylceramide in tissues of patients. Enzyme replacement therapy with recombinant glucocerebrosidase is the mainstay of treatment for Gaucher disease. Here we described the analysis of the disulfide bond pattern, glycosylation site and C-terminal amino acid sequence analysis of recombinant glucocerebrosidase, ISU302. The analysis of the N-glycosylation site was performed by tryptic digestion of the protein and mass measurement before and after enzymatic deglycosylation. We identified four glycosylation sites. It was reported that human acid β-glucocerebrosidase has two disulfides in N-terminal position. We used endoproteinase Glu-C digestion to analyze the N-terminal disulfide bonds. N-terminal and C-terminal fragments were isolated by HPLC and MALDI-TOF MS analysis from endoproteinase Glu-C digests. The isolated N-terminal peptide was chemically modified with or without addition of DTT. This analysis revealed that the first four cysteins located in N-terminal of the protein form two disulfide bonds. C-terminal amino acid sequence was analyzed by Edman sequencing from isolated C-terminal peptide.

J Biomol Tech. 2009 February; 20(1): 54–55.

V52-S2 A New Algorithm for the Analysis of Intact Proteins

Abstract

Currently, the most common algorithm for intact protein mass determination is maximum entropy deconvolution. This method transforms a mass spectrum in m/z units into a mass spectrum containing the zero-charge representation of the intact protein (in Dalton units). Complex data produces false positive “overtone” peaks, which correspond to masses calculated from randomly dispersed peaks from the raw data. Also, if proteins do not perfectly coelute, it is difficult to choose one or more averaged spectra from the chromatogram that produces high quality results for these multiple proteins. Here we describe a new algorithm called “Large Molecule Feature Extraction” (LMFE) for the determination of the masses of large molecules in complex mixtures. This approach first produces extracted ion chromatograms for all peaks in the raw LC/MS data and subsequently groups the peaks with the same retention time and elution profile into “coelution groups”. The peaks within a given coelution group will contain the different charge states of the same protein, which are subsequently grouped together by algebraic charge state deconvolution. While algebraic deconvolution is generally untenable for very complex spectra, the coelution grouping greatly simplifies the peak spectra that are used during algebraic deconvolution making the process much more robust. For a mixture of all E. coli cytosolic proteins, the base peak chromatogram was integrated and peak spectra extracted from each integrated peak. Subsequently, maximum entropy deconvolution was performed on the 90 averaged spectra using a mass range of 6 to 70 kDa. This required 90 minutes of analysis time and resulted in 140 protein compounds. Using LMFE, 682 protein masses were found in 15 minutes, which is almost five times as many compounds in 1/6th the amount of time. Thus, for complex mixtures, LMFE greatly outperforms maximum entropy deconvolution.

J Biomol Tech. 2009 February; 20(1): 55.

V53-S2 Protein N-terminal Sequencing by Mass Spectrometry for Antibody Characterization

Abstract

To develop a mass spectrometry based method which can reliably characterize the N-terminus of antibodies (and proteins in general) in a simple mixture, including determining endogenous N-terminal modifications, and distinguishing minor N-terminal variants. We blocked all primary amines with D3-acetylation in gel separated proteins. This was followed by enzymatic digestion, peptide extraction, primary amine capture (when necessary), LC-MS/MS, data base search, and manual verification of data to identify any free and modified N-termini in the gel band. We started with two standard proteins, one known to have a free, unmodified N-terminus (Bovine serum albumin, BSA) and one that is known to have an acetylated N-terminus (Bovine Carbonic Anhydrase II, CAII) to optimize the labeling and digesting chemistries. The goal of the method is to have every protein N-terminal peptide labeled and that this peptide be the only labeled peptide in the post-digestion mixture. By labeling with heavy (D3)-acetylation we can tell the difference between endogenously and chemically labeled N-termini. We successfully identified both the D3-acetylated N-terminal peptide of BSA and the endogenously H3-acetylated N-terminus of CAII. This method was then applied to the light and heavy chains of a gel separated antibody and data compared to Edman degradation of both chains. Edman degradation identified a single N-terminal species for both the light chain (pyroQ/E removed then SALTQPRSV) and heavy chain (EVQLVESGGG). These results corresponded to the LC-MS/MS analysis of the tryptic peptides of the light chain (pyro-QSALTQPR) and heavy chain (D3-acetylated-EVQLVESGGGLVQPGG-SLR). In addition, MS analysis identified other lower level N-termini in both samples. In the light chain, two other termini were identified, D3-acetyl-SWAQSALTQPR and D3-acetyl-SALTQPR. In the heavy chain, low levels of pyro-EVQLVESGGGLVQPGGSLR were observed. This method readily confirms the N-termini of gel purified antibodies/proteins and can distinguish endogenously modified N-termini from chemically modified N-termini. In addition, he can detect lower abundant N-termini, a clear advantage over Edman sequencing.

J Biomol Tech. 2009 February; 20(1): 55.

V54-S2 Rapid Screening of Protein Aggregates and Protein Impurities

Abstract

Gel filtration (GF) is an excellent tool to acquire information about sizing (identity), purity and specially the multimeric state of a protein of interest. To speed up analysis and keep sample and buffer consumption at minimum, prepacked gel filtration columns of 3 mL (5 mm diameter × 150 mm length) has been used. These columns make it possible to run rapid analysis (6–12 min/run) with minimal sample (4–50 μL) and buffer consumption. Results from a rapid screening of optimal conditions for HIC purification of antibody to minimize dimer and higher aggregate content, assessment of integrity of a recombinant protein and fast purity check of two different Strep-tag II proteins will be presented.

J Biomol Tech. 2009 February; 20(1): 55–56.

V55-S2 Top-down Protein Characterization by ETD/PTR

Abstract

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 allows therefore the facile localization of the PTM. For fragmentation via electron transfer dissociation, 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 the 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.

J Biomol Tech. 2009 February; 20(1): 56.

V56-S2 Top-Down Localization of PEGylation Sites of Therapeutic Peptides by MALDI-ISD

Abstract

In this study, a new approach to localize PEGylation sites is described that utilizes Matrix-Assisted Laser Desorption/Ionization In-Source Decay (MALDI-ISD) mass spectrometry. The sample was a 31mer peptide (3.5 kDa) with blocked N- and C-termini. It was conjugated to 20 kDa PEG for use as a therapeutic peptide (Amgen, disclosed under WO 2007/048026). Peptide and conjugate were prepared with sinapinic acid matrix in a seeded crystallization. The native as well as the conjugated peptide were analyzed using MALDI-ISD in reflector mode on an ultraflex III MALDI-TOF/TOF instrument (Bruker) as described in detail in. The free peptide provided sequence information for large parts of its sequence based on intense y, z+2 and c ions. Without precursor ion selection, the ISD spectrum allowed to confirm its intended structure with only Lys19 exposing a reactive epsilon-amino group as target structure for conjugation of the PEG-aldehyde. The peptide conjugate provided c- and y-ion series that were truncated excluding Lys19 from any sequence readout very clearly, indicating Lys19 as the proper substrate to the conjugation agent. This is remarkable, as an MS/MS analysis with its precursor ion selection was not possible by any method due to PEG oligomers of similar size. MALDI appeared advantageous in analyzing highly heterogeneous PEGylated peptide drugs with its ability to determine the site of PEGylation. No chemical or enzymatic steps were involved, providing for the high throughput that is beneficial for possible routine use.

J Biomol Tech. 2009 February; 20(1): 56.

SR25-S2 Determining the N- and C-terminal Peptides of a Protein with Mass Spectrometry Via Isotopic Labeling

Abstract

We have developed a method to determine the N- and C-terminal peptides of unknown proteins. This method is based on mass spectrometry, chemical modification and differential isotopic labeling. Once identified, the N and C terminal peptides are subjected to MS/MS analysis so that their amino acid sequences can be obtained through de novo sequencing protocols. This method will, when coupled with molecular biology techniques, allow us to discover the gene sequence of an unknown protein, even in the absence of a sequenced genome. Furthermore this approach will be particularly useful as a means to empirically annotate genomes.

J Biomol Tech. 2009 February; 20(1): 56–57.

V57-S2 MALDI Top-Down Sequencing: Calling N- and C-terminal Protein Sequences with High Confidence and Speed

Abstract

This study describes the analysis of the two samples provided by ABRF-ESRG 2009 using Top-Down Sequencing on a MALDI-TOF/TOF (MALDI-TDS). Samples (50 pmol) were prepared using sDHB matrix and run on a MALDI-TOF/TOF (Ultraflex III, Bruker) by in-source decay. Reflector mode ISD spectra (reISD) were directly analyzed by database searching using Mascot. Only in dedicated cases, such as analyzing unique fusion sites, interactive or automatic de-novo sequencing was added to the simple standard procedure. Both proteins (~40 kDa) provided sequence calls from the N-terminus and the C-terminus from the same dataset permitting their identification. Up to 70 sequence calls from either terminus were obtained and, in addition, point mutations/database errors were obtained. c-type ions or y-type ions were used to call protein N-termini and C-termini, respectively. ADH1_YEAST and G3P_RABIT were identified in the study. As reISD spectra allowed monitoring the termini themselves only indirectly by the position of sequence tags on the mass axis, T3-Sequencing was used to directly confirm the protein sequences of the N-termini. MALDI-TDS appears to be very useful for the N-terminal and C-terminal sequencing of proteins. Even a blocked N-terminus does not prevent obtaining an N-terminal sequence as the sequence ladder is not generated by a sequential chemical reaction requiring a free?-amino group, but rather by prompt reactions at the site of electronic excitation in the MALDI ion source. It matches Edman sequencing in some regards and even adds significant analytical capabilities.

J Biomol Tech. 2009 February; 20(1): 57.

V58-S2 Intact Protein N-terminal Sequencing by Mass Spectrometry; Alternatives to Edman Degradation

Abstract

N-terminal sequencing remains a robust and reliable tool to identify proteins, determine expression dynamics in recombinant systems and implement quality control for signal peptide processing and protein degradation. Despite the fact that the traditional Edman technique is very robust and provides de novo capabilities the method suffers from several limitations namely, throughput, sensitivity, cost and specific data interpretation expertise. Mass spectrometry has been utilized as an alternative to Edman sequencing and results have thus far been variable. Most of these mass spec-based methods utilize the bottom-up approach in which the proteins are reduced/alkylated and digested into peptides prior to analysis using the mass spectrometer. However, these labor-intensive methods also have limitations in terms of digestion efficiency and efficient capture of the N-terminal peptides using various isolation methods such as immunoaffinity chromatography. In this work, we present data on N-terminal sequencing using top-down approaches in which the protein is directly infused into the mass spectrometer and fragmented via electron transfer dissociation (ETD). Using the appropriate ion/ion reaction conditions, charge state-reduced species are prominently evident, however a consecutive series of c-ions are obtained by ETD that provide unambiguous sequence determination of the protein N-terminus with minimal sample manipulation. Data from the ABRF ESRG samples will be presented.

J Biomol Tech. 2009 February; 20(1): 57.

V59-S2 Protein N- and C-terminal Sequencing Using Electron Transfer Dissociation Mass Spectrometry

Abstract

Mass spectrometry has drawn more and more attention as an alternative technology to traditional protein N-, as well as C-terminal sequencing. Electron transfer dissociation (ETD) mass spectrometry is particularly advantageous for sequencing applications because ETD is relatively insensible to the size, the amino acid composition and post-translational modifications of proteins, therefore randomly cleaves protein or peptide backbone bonds. ETD of intact proteins performs with high efficiency, generating very informative, yet extremely complex spectra which contain highly charged product ions that are difficult, or even impossible to resolve at unit resolution. ETD was recently implemented in a hybrid linear ion trap—orbitrap mass spectrometer facilitating the analysis of intact proteins using ETD with its high resolution and accurate mass capabilities. For unit resolution instruments, proton transfer reaction (PTR) following ETD was developed to reduce spectral complexity. PTR removes protons from multiply charged product ions, generating a simplified spectrum that contains product ions of resolvable charge states at unit resolution. In this study, ETD was applied to protein N- and C-terminal sequencing both in hybrid linear ion trap and in unit-resolution linear trap. ETD with accurate mass and high resolution was employed to study optimized reaction conditions for protein N- and C-terminal sequencing. Our results using intact proteins ranging in size from 8 kDa to 46 kDa indicated that optimized ETD reaction time for N- and C-terminal sequence coverage was longer than that for maximum protein sequence coverage. Extended ETD reaction leads to near complete N- and C- terminal sequence covering 30 to 50 amino acids while the overall protein sequence coverage decreased. The optimized ratio of analyte to ETD reagent was also investigated. In a unit-resolution instrument, PTR following ETD of intact proteins up to 30 kDa increased N- and C- terminal sequence coverage when compared to ETD alone.

J Biomol Tech. 2009 February; 20(1): 57.

V60-S2 Straight Protein QC by Benchtop MALDI-TOF Top-Down Sequencing

Abstract

We describe for the first time the straight identification/sequence validation of intact proteins by MALDI top-down sequencing with a benchtop MALDI-TOF. A routine application for protein sequencers is the validation of proper protein structures, in particular from recombinant work. We show that a small benchtop MALDI can actually provide meaningful N- and C-terminal sequence information from undigested proteins. Protein samples (ABRF-ESRG 2009 study samples 1 and 2) were mixed in sDHB or SA matrix and approx. 20 pmol were applied to the MALDI steel target. A benchtop MALDI-TOF (micro-flex LRF, Bruker) with N2 laser was used in reflector mode to acquire in-source decay spectra. Protein identification by standard MS/MS search using Mascot 2.2 (Matrix Science). The proteins in the study (~40 kDa) were identified as ADH1-YEAST and as G3P_RABIT. The latter protein was identified as fusion protein with an N-terminal Histag beta-Gal Vector. Uniquely in this approach, a single reflector mode ISD spectrum of the undigested protein as provided by the organizers was used for the analysis of each sample. Typically the near terminal fragments were monitored by ISD (residues: ~10–50 from either terminus). However, a match of the retrieved protein sequence to the ISD dataset confirmed the proper sequence of the terminal residues although they were not observed on a residue by residue base. A straightforward, quick and cheap approach to QC isolated proteins—recombinant and natural alike—was described and demonstrated. The approach utilized a benchtop MALDI-TOF and standard sequence database searching. For QC type problems with existing sequence candidates, the sequencing requirement would be dwarfed to merely match the sequence and the experimental ISD fragments—a matter of seconds.

J Biomol Tech. 2009 February; 20(1): 58.

V61-S2 MALDI-ISD-FTICR MS for Rapid Top-Down Analysis of Protein Termini

Abstract

For the first time, MALDI-ISD-FTMS MS was used for Top-Down protein characterization. Fast and reliable assignment of N- as well as C-terminal protein sequences is an increasingly important task in protein biochemistry for, e.g., quality control of recombinant proteins, and even within the field of proteomics. Here, we introduce MALDI-Top-Down-Sequencing on an FTMS instrument, on which so far only the ESI-based ECD fragmentation has been introduced as Top-Down analysis method. Matrix-Assisted Laser Desorption/Ionization In-Source Decay (MALDI-ISD) has been shown to efficiently produce c- and z+2-type ions for rapid analysis of both termini of a protein. The workflow consisted of MALDI target preparation, MALDI-ISD mass measurement at slightly elevated laser fluence and subsequent data analysis. Time-consuming digests or LC separations were not involved. Database searches (Mascot) were triggered with low ppm mass tolerances providing ADH1 and G3P identities with respective sequence calls from the ABRF-ESRG 2009 study samples. Using Fourier Transform Ion Cylclotron Resonance Mass Spectrometry (FTMS) for protein identification and assignment of protein termini provided unambiguous assignment of C- and N-terminal sequences. Uniquely, due to the low ppm mass tolerances used in this approach, even Q. vs. K were safely assigned from undigested proteins. MALDI-ISD-FTMS is a new technique complementary to classical ESI-ECD, which rapidly identifies proteins with high confidence calling on their terminal sequences even from 40 kDa and larger proteins.

J Biomol Tech. 2009 February; 20(1): 58.

SR26-S2 Resolution and Quantification of Protein Isoforms in Complex Genomes

Abstract

One of the difficulties in analyzing the results of shotgun proteomics experiments is the differentiation of protein isoforms from the identified peptides. This problem arises when the sequence database of the organism of study contains many similar (but unique) proteins, either because the genome contains related families of genes or because the database was constructed from different sequenced isolates and thus contains many gene isoforms. In this work we demonstrate how to effectively resolve protein isoforms in complex proteomic analyses by combining molecular weight information, protein grouping, peptide filtering and set logic. To illustrate this approach, a proteomic analysis was performed on membrane proteins isolated from Trypanosoma cruzi (T. cruzi, blood stream form trypomastigotes. T. cruzi is the causative agent of human Chagas disease and with over 30% of its genome being comprised multi-copy gene familes, the proteome is highly redundant and difficult to discern by traditional proteomic methos. Using this approach, 2601 total proteins were identified, including validation of expression for greater than 700 large gene family members.

J Biomol Tech. 2009 February; 20(1): 58.

SR27-S2 N-Terminal Enrichment—Developing a Protocol to Detect Specific Proteolytic Fragments

Abstract

Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a “bottom-up” strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics. We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56-Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and ~95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo aceylated peptides from chemically tagged peptides. In an initial experiment using mouse plasma, we were able to identify >300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases.

J Biomol Tech. 2009 February; 20(1): 58–59.

SR28-S2 IVICAT for the Masses: An Improved Technique for Permethylation of Peptides

Abstract

To determine the levels of post-translational modifications of parasite proteins, in particular those of Toxoplasma gondii and Cryptosporidium parva, we needed a quantitative technique that would allow comparison of the amounts of acetylated versus mono-, di and tri-methylated lysines in histones and other proteins. Recently, several laboratories determined relative quantities of peptides using iodomethane and deuterated iodomethane and showed derivitization of peptides to give a trimethylated amino terminus and/or trimethylated lysines. These trimethylated peptides showed increased signal intensity in the mass spectrometer. The power of this approach, in vacuo isotope coded alkylation technique (IVICAT), is offset by its technical difficulty: a hot torch must be used to seal the glass sample tube while it is under vacuum and cooled by liquid nitrogen. Also, the pH of the peptide solution has to be carefully adjusted prior to lyophilization. Using model peptides and proteins, we have simplified and improved the technique by employing a glass screw cap vial equipped with an inert cap and valve. A standard lyophilizer served as the vacuum source. We were able to use lower amounts of peptides and react multiple peptide samples within the same vial. We permethylated with high recovery lyophilized peptides, rotary concentrated peptides and peptides bound to reverse phase chromatography media packed into pipette tips. Several aspects of the chemistry and mass spectrometry of the permethylated peptides will be presented. Our improved technique makes IVICAT accessible to most proteomics laboratories and may be used to provide more complete sequence coverage of proteins and proteomes in addition to quantitation. This work was supported by NIH/NIAID Contract HSN266200400054C.

J Biomol Tech. 2009 February; 20(1): 59.

SR29-S2 Searching for Efficient and High-Throughput Alternatives for Essential Sample Preparation Techniques in Mass-Spectrometry-Based Functional Proteomics

Abstract

Good control, efficiency, and reproducibility of protein extraction from cells and tissues are essential for diverse biological and basic research applications. Effective and specific proteolytic digestion of proteins prior to mass spectrometry (MS) analysis is one of the fundamental techniques most commonly used in any proteomics laboratory. However, neither routine procedures for cell and tissue lysis nor for in-solution and in-gel protein digestion have been significantly altered in common practice for over a decade. Here, we have tested and optimized several alternative techniques for preparing lysates of mammalian cells, as well as in-solution and in-gel enzymatic digestion compatible with downstream qualitative and quantitative MS-based proteomics applications. Specifically, we used alternating hydrostatic pressure (pressure cycling technology, or PCT) and specialized organic solvents for disruption of cells, micelles and membrane fragments and efficient protein recovery from cultured cells. Using high performance LC-MS analysis, we have tested the role of pressurization, organic solvents, chaotropic agents, reducing reagents, enzyme/substrate ratios, and incubation time on efficiency, selectivity, and throughput of proteolytic digestion. The tests were performed using a mixture of protein standards and the most effective conditions were applied to HepG2 cell lysates. The optimized conditions for pressure-assisted in-solution digestion were adapted to in-gel digestion of 1D-PAGE fractionated HepG2 lysates and protein identification results were compared to those acquired using optimized digestion conducted at atmospheric pressure. Application of PCT resulted in significant improvement of throughput and reproducibility of sample preparation for proteomic analyses. Superior extraction rate for cytosolic and membrane associated proteins, as well as for proteins related to many other gene ontology terms, biological pathways and interaction networks, were observed at pressure-assisted sample preparation.

J Biomol Tech. 2009 February; 20(1): 59.

V62-S2 Comparison of Cellular Phosphoprotein Responses with High Quantitative Confidence Using Pulsed High Pressure Proteolytic Digestion and Automated Phosphopeptide Enrichment

Abstract

The analysis of the regulatory elements and dynamics of cellular signaling events present multiple challenges. Proteomic approaches to understanding signal transduction provide an unbiased and highly comprehensive analysis of global events. However, phosphoprotein expression profiling is often limited by the technical difficulty and reproducibility associated with sample preparation. We have treated A431 and HeLa cells with growth factors in the presence and absence of kinase inhibitors or siRNA. Protein from these cells was extracted and digested (with multiple proteases) using a novel pulsed high-pressure lysis apparatus. Peptides were then labeled with tandem mass tags (TMT), desalted, and then fractionated using strong cation exchange (SCX) chromatography. Automated phosphopeptide enrichment was performed using magnetic TiO2 beads. We have used these protocol enhancements to identify, quantify, and profile phosphorylation sites across technical replicates with good reproducibility. These results demonstrate the ability to quantitatively profile complex phosphoprotein mixtures in different cancer types with statistical confidence using a combination of protocol and reagent enhancements.

J Biomol Tech. 2009 February; 20(1): 60.

V63-S2 A Multi-Laboratory Study Assessing Reproducibility of a 2D-DIGE Differential Proteomic Experiment

Abstract

Although 2DE-electrophoresis has been long used to study differential proteomics, its reproducibility has been always a major concern. In recent years, different methodological improvements have contributed to more robust 2DE workflows: use of immobilized IEF strips, fluorescence based difference gel electrophoresis (DIGE), new software tools, etc. In order to assess the reproducibility of 2DE experiments across laboratories, we set up a multi-laboratory study, performed at 11 laboratories of the ProteoRed network (Spanish network of proteomics facilities). All participating labs received two protein extracts, prepared from cultured human adenocarcinoma MDA-MB-468 cells, treated or not with 50 ng/mL EGF (Epidermal Growth Factor) for 24 h. Differential analysis was performed by a four-gel 2D-DIGE experiment, using four technical replicates of each sample, with Cy dye swapping. Strictly defined 2DE conditions were followed by all labs. Each lab selected the 30 spots presenting the highest fold variations (with p<0.05), and attempted MS protein identification.

The results demonstrate a very good within lab and across lab reproducibility. Within labs, 75–85% detected spots present %CV < 10%, and 40–60 %CV < 5%. Across all labs, around 60% and 15% of spots show %CV < 10% and <5%, respectively. Selection of differentially expressed spots shows good reproducibility across labs, although there is a certain degree of subjectivity in the selection, as each lab applied its own filtering criteria. Overall, 24 spots were ranked among the top-30 by at least three labs, and 14 by at least four. MS protein identification was, on average, 60% successful, with 22 spots identified by at least three different labs. In those cases, identical gel locations corresponded to the same protein Id. In conclusion, the results of the study show the robustness of the methodology used, and demonstrate the feasibility of across lab validation schemes, pointing towards development of inter-lab QC strategies for proteomic research.

J Biomol Tech. 2009 February; 20(1): 60–61.

V64-S2 Enrichment with High Specificity of Phosphopeptides at Low pH Prior to Mass Spectrometry Analysis

Abstract

A new product will be presented designed for single use small-scale phosphopeptide enrichment. The chromatographic mechanism is based on IMAC (immobilized metal ion affinity chromatography) and the beads are precharged with Fe3+. The binding of phosphopeptides to is pH-dependent and the advantage of performing the binding at an extremely low pH has been utilized for a high-purity and reproducible enrichment of phosphopeptides. Different types of protein samples were used in the study. First, a model peptide from trypsin digested bovine β-casein, spiked into a background of albumin tryptic fragments was tested. Secondly, different complex samples, such as cell extract from S. cerevisiae, HeLa cells and also E. coli extract spiked with model proteins were analyzed. Each cell extract was diluted in 50 mM glycin-HCl, 50% acetonitrile, pH 2.0. After tryptic cleavage the sample was loaded and captured on microspin columns packed with the new IMAC medium and eluted with 1% phosphoric acid, 50% acetonitrile, pH 1.7. All eluates were analyzed with mass spectrometry. To avoid the need for methylesterfication of acidic protein/peptides, several experiments were performed to optimize the binding of the peptides at low pH. According to the mass spectrometry analysis, a high signal/noise ratio was obtained for the phosphopeptides in a complex sample. The spiked model proteins were identified in the clarified E. Coli extract and seventeen phosphopeptides were identified with high significance (p < 0.001). For the extract, forty-one phosphopeptides encoding thirty-eight proteins were detected with high significance (p < 0.001) in the eluted material. Data from the study shows that during low pH conditions in sample binding and elution the new Fe3+ precharged IMAC medium has a high specificity for phosphopeptides and can be used for enrichment of phosphopeptides from complex samples with a high level of reproducibility.

J Biomol Tech. 2009 February; 20(1): 60–61.

V65-S2 Parallel Isoelectric Focusing of Tryptic Peptides by the Digital ProteomeChip

Abstract

MS-based proteomics of complex samples has evolved from single spot analysis of 2D gels to high throughput methods such as MudPit, where thousands of proteins are identified from a single sample. For these techniques, fractionation methods are usually employed to reduce the complexity of the sample prior to LCMS thus providing an increase in protein coverage. The objective of this study is to investigate the use the parallel isoelectric focusing of tryptic peptides as a sample prep method for LCMS proteomics. Parallel isoelectric focusing using the digital ProteomeChip dPC™ was used to separate tryptic peptides from a complex mix in 30–60 minutes. The anodic and cathodic buffers contained octoglucopyranoside, added for maximal resolution and solubility during the separation. The peptide amount may range between 20 and 400 μg. After pI separation, the peptides were passively are eluted from the gel plugs. We show that this sample matrix is directly compatible with ESI MS. Also, fluorescent labeled peptides were used to demonstrate reproducible pI separation between multiple dPC lots. In conclusion, we demonstrate that parallel isoelectric focusing of peptides provides a rapid, reproducible and easy to use pI based separation for proteomic applications.

J Biomol Tech. 2009 February; 20(1): 61.

SR30-S2 Using IEF Fractionation of Tryptic Peptides to Reduce False Protein Identifications and to Minimize Estimates of False Discovery Rates in Proteomics Experiments

Abstract

Searching massive protein sequence databases with large sets of tandem mass spectrometry data invariably leads to false peptide/protein identifications. Estimates of the false discovery rate (FDR) associated with an experiment can be made by searching suitable decoy databases. However, such an approach cannot be used to flag any of the putatively identified peptides as likely false positives. A strategy that can be employed to deal more effectively with the problem of false discovery is to determine additional physical constants of the peptides found within the samples. These can then be used as supplementary filters for the data. Here we show that a comparison between the peptide isoelectric point (pI), determined experimentally by using “off-gel” IEF as a pre-fractionation step prior to LC MS/MS analysis, and the pI calculated for all putative peptide hits can be used to flag specific peptide identifications as “likely to be false.” A similar comparison for putative hits to a decoy database is shown to reduce the estimated FDR by factor of three in samples isolated from tomato fruit. LC-MALDI-MS/MS is recommended to analyze the IEF fractions, particularly when the isoelectric focusing is run at standard ampholyte concentrations. LC-ESI-MS/MS can also be used, providing the fractionation is run at greatly reduced ampholyte concentrations. Using reduced ampholyte concentrations reduces the correlation between the measured pH of the fractions and the published pI range of the IPG strips. Nevertheless, it is shown that reasonable estimates of the isoelectric points of the peptides found in the fractions can be made from the published pI range of the IPG strip. Since peptide IEF fractionation provides an experimentally determined estimate of the peptides’ pI it is a superior 1st dimension separation strategy to SCX chromatography in shotgun proteomics applications.

J Biomol Tech. 2009 February; 20(1): 61.

SR31-S2 Proteome Changes between Genetically Related Helicobacter Pylori That Differ in Carcinogenic Potential Revealed by DIGE/MS

Abstract

Helicobacter pylori represents the strongest known risk factor for gastric adenocarcinoma, yet only a fraction of infected persons develop cancer. We use DIGE/MS to identify differentially-expressed proteins between a non-carcinogenic H. pylori strain and its carcinogenic derivative. Independent quadruplicate samples were pre-fractionated and analyzed by pH 4–7 and pH 7–11 DIGE using 8-gel sets coordinated by a Cy2-labeled sample mixture to provide every resolved form with a unique internal standard for cross-gel normalization/quantification. 842 resolved protein features (including charged isoforms) were matched across the resulting 16 pH 4–7 proteome maps, for which Principal Component Analysis (PCA) clearly indicated that the major source of variation (PC1, 80.3%) distinguished the cytoplasmic and membrane samples, as expected. An additional 5.2% of variance (PC2) separated the carcinogenic strain 7.13 from the non-carcinogenic strain B128. Similar results were obtained for an additional 300 protein features resolved in the pH 7–11 range. Unsupervised Hierarchical Clustering similarly demonstrated high reproducibility between independent replicate samples and an extremely low level of background noise (technical variation), and highlighted a discrete subset of protein expression changes representing candidate proteins potentially involved in carcinogenesis. Subsequent MS-based protein identification indicated multiple classes of proteins that displayed strain-specific differences, including oxidative stress response, evasion of the immune system, and bacterial replication. One notable difference was found for the FlaA flagellar protein. Peptide mass mapping clearly demonstrated a unique peptide ion at m/z 732.38 that was specific to FlaA from strain B128. The resulting TOF/TOF fragmentation pattern was consistent with the sequence GC(296) LNLR, whereas an arginine is normally present at position 296. This residue maps to a region predicted helical region, and mobility and ultrastructural analysis indicated that the R-to-C mutation correlated with decreased motility and increased flagellar adherence, consistent with a motility-based model of carcinogenic potential.

J Biomol Tech. 2009 February; 20(1): 61–62.

SR32-S2 Proteomic Comparisons of Differentiating Embryonic Stem Cells, an In Vitro Model of Embryogenesis

Abstract

Embryonic stem cells, early primitive ectoderm-like cells, and embryoid bodies may be used as an in vitro model of embryogenesis. These cells are representative of the in vivo 4.5 d.p.c. inner cell mass, the 5.5 d.p.c. primitive ectoderm, and the 6–8 d.p.c differentiated embryo, respectively. Here, we use progressively differentiated embryonic stem cells as an in vitro model of embryogenesis. We explore protein regulation of these differentiating cells using shotgun proteomics and spectral counts. Peptides identified from each population were clustered to proteins at a 1% FDR. A total of 2911 proteins (1013 protein homology groups) were identified for the ES proteome, 1852 proteins (568 protein homology groups) for the EPL proteome, and 3328 proteins (1038 protein groups) were identified in the embryoid body proteome. Uniquely identified proteins were grouped according to comparative and single proteomes, forming a total of 1509 proteins shared across all three proteomes. Proteins shared between cell states represented over 50% of the protein population. These proteins formed four comparative proteome groups consisting of proteins shared between ES and EPL, ES and EB, EPL and EB, and between all three proteomes (ES, EPL, and EB). Average spectral counts were used as a semi-quantitative measure of protein abundancy for comparative proteomes. Proteins typically used as loading controls during western blotting were used as control proteins to evaluate protein normalization techniques. For the general population of identified proteins, variations in the %RSD ranged from 1% RSD to 151% RSD. Ingenuity software was used to investigate functionality between comparative proteomes. Top functions were those identified at a >95% confidence level. Some of these functions were cell growth (ES/EPL), RNA post transcriptional modification (ES/EB), and cell movement (EPL/EB). Regulation of proteins in these identified functional groups could be correlated to embryogenetic events.

J Biomol Tech. 2009 February; 20(1): 62.

SR33-S2 Quantitative Proteomics in Biomedical Research

Abstract

Proteomics and mass spectrometry have provided unprecedented tools for fast, accurate, high throughput biomolecular separation and characterization. Studying at the protein level allows researchers to investigate how proteins, their dynamics and modifications affect cellular processes and how cellular processes and the environment affect proteins. Here we present our capabilities in quantitative proteomics and MS analysis. The tools include gel-based 2D-DIGE (two-dimentional difference gel electrophoresis) and gel-free iTRAQ (isobaric tags for relative and absolute quantitation) technologies. Along with our capacity of separating proteins and characterizing differential protein expression, we have a suite of mass spectrometers available for biomedical research, including a 4700 TOF-TOF Proteomics Analyzer, a quadrupole/time-of-flight (QSTAR XL), and a quadrupole-linear ion-trap (4000 QTRAP). These instruments are mainly used for protein identification, posttranslational modification characterization and protein expression analysis. In addition to 2D-DIGE and iTRAQ, we have recently explored the multiple reaction monitoring (MRM)-based protein quantification. Our facility is fully set up to synthesize and purify peptides. With synthetic standard peptides, MRM technology, which is a gold standard for drug analysis, allows sensitive and absolute quantification of proteins.

J Biomol Tech. 2009 February; 20(1): 62.

SR49-S2 Dynamics of the MAPK Insulin Signaling Pathway Using a Label-Free Platform Based on MS/MS Average TIC

Abstract

Large-scale proteomics experiments similar to those first introduced by Gavin et. al. are needed in order to interrogate protein-protein interactions in cellular signaling pathways. We approached this question by performing TAP-MS experiments on 15 key nodal bait proteins in the MAPK pathway in drosophila SR+ cells with and without insulin and EGF (Spitz) stimulation. Data dependent LC/MS/MS experiments were run using a Proxeon EasynLC coupled to a Thermo LTQ-Orbitrap XL for at least two replicates of each bait condition for a total of 94 LC/MS/MS experiments including TAP vector controls. The data was searched against the reversed Flybase protein database due to its completeness using Sequest within Thermo’s Proteomics Browser Software suite. In order to quantify changes in protein levels between bait conditions, we developed a software suite called Naked Quant v1.0 based on the “Spectral TIC” approach that averages the TIC values from each identified MS/MS spectrum (spectral count) per protein identified. The method was previously compared to SILAC and spectral counting in a proteomics screen (Asara et. al., Proteomics, 2008). The software utilizes several features including protein grouping across experiments, normalization as well as fold change and ratio calculations based on spectral counts, TIC sum and TIC average. From the output, different networks were assembled based on the dynamic average TIC signal change using several different criteria including simple fold changes between basal and stimulated conditions using common identified proteins and pair wise changes in signal between basal and stimulated conditions where at least two or more proteins need to change together. The network revealed many canonical interactions and several novel interactions in the MAPK signaling pathway. Several of these novel interactions have been verified biochemically and correlate with a previously published RNAi screen using pERK as the readout under basal and insulin treatment (Friedman and Perrimon, Nature, 2007). These data show that novel interactions in signaling pathways through protein-protein interaction studies can be effective using label-free mass spectrometry approaches.

J Biomol Tech. 2009 February; 20(1): 62–63.

V66-S2 ICPL 4-plex: Isotopic Protein Labeling for Quantitative Protein Analysis by nano-LC-MALDI-TOF/TOF

Abstract

Mass spectrometry based quantitative proteomics has become an important component of biological/clinical research. Quantitative protein analysis requires methods that are able to provide accurate/reproducible differential expression values for proteins in two or more biological samples. Stable isotopic labeling with LC-MALDI-MS/MS-analysis has emerged as a powerful tool to measure the relative quantitative differences with high accuracy. The new 4-plex isotope coded protein labeling (4-plex ICPL) approach is based on the labeling of intact proteins by amine-specific ICPL reagents (Bruker). The labeling step is typically applied prior to protein–pre-fractionation/separation steps and prior to protein digestion. It, therefore, uniquely supports Top-Down proteomics studies involving, e.g., SDS-PAGE protein separations. Various protein mixtures with different complexity were subjected to the new 4-plex ICPL reagent. Four different low complex protein samples (10 proteins) were successfully analyzed by nano-LC-MALDI-TOF/TOF analysis. After the MS analysis of the complete LC-run peptide peaks with appropriate labeling were determined by the processing software and quantification ratios were calculated using median statistics. For identification of the regulated peptides, the most abundant peptide was automatically MS/MS analyzed. The WARP-LC 1.2 software (Bruker) was used for the quantification of the 4-plex ICPL-labeled proteins, which is used for the quantification of iTRAQ and SILAC labeled peptides and proteins as well. Due to the common request for the determination of quantitative differences among >2 biological samples, the 4-plex ICPL approach was also subjected to the quantification of several target proteins present in the more complex E. Coli proteome.

J Biomol Tech. 2009 February; 20(1): 63.

V67-S2 Ruggedness of Nanobore LCMS for Qualitative and Quantitative Biomarker Analysis Using an Automated Emitter Position and Rinsing System

Abstract

Nanobore LC-MS is a technique used for qualitative analysis of complex proteomes extracted from biological matrices including plasma, cell lysates and tissue extracts. As the need for qualitative nanobore LC-MS shifts to quantitative analyses, system robustness is critical. Complex mixture analysis presents significant challenges, particularly when operating under nanoflow conditions. The lifetime of nanobore components (traps, column, emitters) can be significantly compromised when analyzing crude samples extracted from biological matrices. A systematic investigation of contamination resulting from the analysis of plasma was conducted. Contamination from the thermal degradation of biochemicals (proteins, peptides, lipids) is a factor because the nanospray emitter is typically in close proximity to the heated atmospheric pressure inlet of the mass spectrometer. The contamination manifests as a coating on the exterior surface of the emitter. Such contamination can change the surface wetting properties of the mobile phase, negatively impacting spray stability. An automated digital control positioning system enables the exterior of the emitter to be washed with a suitable solvent at regular intervals, minimizing the buildup of contamination on the exterior of the emitter. The performance improvements of washing the emitter were tested by analyzing samples derived from canine plasma samples spiked with two quantitative controls, one a small molecule and the other, a peptide. The recovery of these two standards was monitored throughout sequential injections and plotted. Improved robustness as a result of regular emitter rinsing is under long-term investigation.

J Biomol Tech. 2009 February; 20(1): 63–64.

V68-S2 Intelligent Use of Retention Time for Higher Order Multiple Reaction Monitoring Multiplexing—Scheduled MRM™ Algorithm

Abstract

Targeted peptide quantitation is a rapidly growing application within proteomics mass spectrometry due to its widespread utility in biomarker verification, protein/peptide confirmation and characterization, as well as pathway mapping. The utility of Multiple Reaction Monitoring (MRM) on triple quadrupole based MS systems for these studies is currently an active area of investigation, driven by the well known sensitivity and selectivity attributes of this type of MS approach. As more extensive protein panels need to be monitored in a targeted way across multiple samples, higher MRM multiplexing is becoming essential for throughput. The need for rapid assay development, higher multiplexing and more robust assays are some of the key challenges. In this work, the unique combination of triple quadrupole and ion trapping capabilities of the hybrid triple quadrupole—linear ion trap mass spectrometer (QTRAP® System) has been utilized to create hundreds of high quality, specific MRM transitions for multiple peptides to many plasma proteins. Human plasma was separated by reversed-phase HPLC on an Eksigent Ultra nanoLC System. MRM analysis was performed using the Nano-Spray® source on an Applied Biosystems/MDS Analytical Technologies QTRAP® 5500system. By using the MRM-Pilot™ Software coupled with the MIDAS™ workflow, all MRM transitions to plasma proteins were designed. Using MRMPilot, iterative MRM analysis was performed which provided rapid refinement of MRM parameters without requiring synthetic peptides. Intelligent use of retention time using a new acquisition software (Scheduled MRM) enabled many more MRM transitions to be included in a single acquisition method, while maintaining good peak area reproducibility. Here, using an MRM method created with Scheduled MRM™, 784 MRM transitions to 98 proteins in human plasma was monitored in a single analysis. Ten replicate analysis was performed to test for reproducibility. The plasma protein peptides were labeled with mTRAQ™ reagent, so four MRM transitions to the light version and four to the heavy version of each peptide were monitored. In spite of a large number of MRMs monitored in a single run, over 90% of peptide peak areas and ratios (light over heavy) had reproducibility better than 5% CV. This was made possible by using Scheduled MRM™ algorithm which separated the MRMs by retention time and thus sampled only a portion of the MRMs over a specific time window versus sampling all 784 MRMs every cycle over the whole gradient. The usage of MRMPilot and Scheduled MRM has automated and simplified the complete workflow of taking discovery data, to refinement of MRMs and to creation of a method containing the final list of a large numbers of MRMs along with its elution time. These processes would be very laborious and time comsuming if done otherwise or maually. In addition, it would not be feasible to sample a large number of MRMs in one analysis without sacrificing the MRM peak quality which would affect the quantitation accuracy. Presented here are the details of the workflow of using MRMPilot and Scheduled MRM™ to generate a large list of MRMs and its results are discussed.

J Biomol Tech. 2009 February; 20(1): 64.

V69-S2 Quantitative Differentiation of Secreted Proteins from Mycobacterial Strains

Abstract

In microbial pathogenesis, virulence originates from multiple sources; genetic content, changes in phylogeny, and differences in protein localization. Mycobacterium tuberculosis is an acid-fast bacillus responsible for 1.75 million deaths annually. A major aspect of mycobacterial virulence includes the secretion of virulence proteins. The precise role of these exported factors in virulence is unclear. Because M. tuberculosis is a slow-growing mycobacteria, other, more rapidly growing pathogenic mycobacteria, including M. marinum are often used as models for studying M. tuberculosis. Here, we employed a unique combination of iTRAQ and mTRAQ chemistries to label the secreted proteomes from M. tuberculosis and M. marinum. The goal of this study was to identify quantitatively specific differences in the secreted proteomes. These differences determined by isobaric labeling were then further characterized and defined by stable isotopic labeling of substrates determined to be different between different strains of WT tuberculosis and related model-organisms. We were able to determine that many known virulence factors exhibited distinct quantitative changes between different strains. The output of this work is to create a method that would enable the quantitative differentiation from clinical isolates of tuberculosis. The results of this screen and the correlation of substrate quantitation with virulence will be discussed.

J Biomol Tech. 2009 February; 20(1): 64.

V70-S2 Novel Protein Expression Assays Using qPCR for the Detection and Relative Quantification of Protein Markers in Human Embryonic Stem Cells

Abstract

Quantitative PCR (qPCR) has revolutionized the characterization of nucleic acids in cells, and several classes of cellular nucleic acids are routinely analyzed by qPCR assays, including genomic DNA, mRNA and microRNAs. Proximity ligation assay (PLA) technology extends qPCR applications now to the detection of cellular proteins through the amplification of a surrogate DNA template. PLA is a three-step process that involves, (1) binding of paired antibody-oligonucleotide probes to a protein target in biological samples, (2) templated ligation of the oligonucleotides in proximity, and (3) qPCR detection. We have optimized this technique for crude cell lysates utilizing a simple, onestep sample lysis approach to release all classes of proteins, and combined it with gold standard TaqMan® chemistry to create a highly sensitive and specific process for measuring protein expression in small samples. One application of this assay is the detection and relative quantification of markers in pluripotent and differentiated stem cells. Stem cell characterization typically relies on determining the presence and amount of stage specific protein markers such as OCT4, NANOG, SOX2, and LIN28. Protein expression results confirm cell-stage specific changes in protein expression of these key stem cell markers, and the data can be directly compared with published mRNA expression profiles for the same cell lines. We have engaged a number of researchers as test sites for these assays and are gathering input and feedback. Our findings illustrate how this new assay system expands the scope of qPCR to protein detection and quantification, an important area of cell biology.

J Biomol Tech. 2009 February; 20(1): 64–65.

SR34-S2 Identification of a Novel Pathway in Stimulated Human T-Cells That Drives Macrophage Activation and Cytokine Production

Abstract

The NAD-dependent deacetylase SirT1 regulates lipid and glucose metabolism in liver and increased SirT1 activity in caloric-restricted models has been linked to extended life span in several species. Deacetylation of transcription factors and co-modulators, such as p53, NF-κB, C/EPBβ and PGC-1α, allows SirT1 to sense and regulate energy levels. The SirT family can also deacetylate mitochondrial proteins, suggesting that posttranslational modification by sirtuins may have global effects on energy metabolism especially gluconeogenesis and lipogenesis. Livers from mice fed either control or a high fat (HF) diet (45 kcal% as fat, 12 wks) were harvested and homogenized. SirT1 levels were measured in whole cell lysates by Western blot. Proteins modified by lysine acetylation were immunoprecipitated with anti-acetyllysine antibody and subsequently separated by one dimensional gel electrophoresis. Bands showing differential staining between the control and HF fed mice were excised and proteins were digested with trypsin. Tandem mass spectrometry using an Agilent Ultra quadrupole ion trap generated product ion spectra that were searched with SpectrumMill against the SwissProt database. The levels and acetylation of identified proteins were validated by immunoprecipitation and Western blotting. Mice on the HF diet were obese, with fatty livers and reduced SirT1 activity as assessed by the NAD+/NADH ratio. SirT1 protein expression levels did not significantly change, however. Acetylation of a subset of the proteins identified, such as carbamoyl-phosphate synthase, uricase, pyruvate carboxylase and ATP synthase, has been previously reported. Interestingly, peroxiredoxin, catalase, and Hsp70, proteins involved in redox and the stress response, were hyperacetylated in the livers of obese mice. We postulate that modification of those proteins could influence the ability of obese mice to modulate oxidative stress, gluconeogenesis and lipogenesis. Globally surveying lysine acetylated proteins using immunoprecipitation and gel electrophoresis/tandem mass spectrometry provides insights into how obesity impacts liver metabolism.

J Biomol Tech. 2009 February; 20(1): 65.

SR35-S2 Proteomic Analyses of Pancreatic Cyst Fluids

Abstract

There are currently no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. Proteomic analyses were performed to explore pancreatic cyst fluids to yield effective diagnostic biomarkers. We have prospectively recruited 20 research participants and prepared their pancreatic cyst fluids specifically for proteomic analyses. Proteomic approaches applied were: (1) MALDI-TOF (matrix-assisted laser-desorption-ionization time-of-flight) mass spectrometry peptidomics with LC/MS/MS (HPLC-tandem mass spectrometry) protein identification. (2) 2D gel electrophoresis. (3) GeLC/MS/MS (tryptic digestion of proteins fractionated by SDS-PAGE and identified by LC/MS/MS). Sequencing of over 350 free peptides showed that exopeptidase activities rendered peptidomics of cyst fluids unreliable; protein nicking by proteases in the cyst fluids produced hundreds of protein spots from the major proteins, making 2D gel proteomics unmanageable; GeLC/MS/MS revealed a panel of potential biomarker proteins that correlated with CEA (carcinoembryonic antigen). Two homologs of amylase, solubilized molecules of four mucins, four solubilized CEACAMs (CEA-related cell adhesion molecules), and four S100 homologs, may be candidate biomarkers to facilitate future pancreatic cyst diagnosis and risk-stratification. This approach required less than 40 microliters of cyst fluid per sample, offering the possibility to analyze cysts smaller than 1-cm diameter.

J Biomol Tech. 2009 February; 20(1): 65.

SR36-S2 High Mobility Group Box 1 (HMGB1) In Eosinophil Activation

Abstract

Eosinophils have been implicated in allergic inflammation and certain parasitic and viral infections. Our proteomic studies of non-activated and activated eosinophils identified the expression and release of HMGB1. HMGB1 plays a prominent role in immunoregulatory cell activation including granulocyte activation. Our experiments were designed to determine whether eosinophil-derived HMGB1 contributes to the autocrine activation of human peripheral blood eosinophils. HMGB1-presence in eosinophils was determined using western blotting and two-dimensional gel electrophoresis. Pro-Q Diamond phosphoprotein stain and Sypro Ruby protein stain were used for detection of phosphoproteins or proteins, respectively. Cell survival rates and expression of CD69 were determined after stimulation of eosinophils with GM-CSF, rHMGB1, and various HMGB1 inhibitors. Eosinophils stimulated with either GM-CSF, rHMGB1, and eosinophil-derived HMGB1 showed significantly higher viability and expression of CD69. Pre-treating eosinophils with glycyrrhizin, a specific inhibitor of HMGB1 activity, partially inhibited prolongation of survival as well as upregulation of CD69. This upregulation was significantly inhibited by treatment with glycyrrhizin or anti-RAGE2 antibody. The phosphoproteomic profile of HMGB1 stimulated eosinophils also changed. The phosphoproteomic pattern and the proteomic pattern of eosinophils treated with HMGB1 and GM-CSF vary as compared to control cells and compared to each other. Our studies demonstrate the autocrine activation of eosinophils through HMGB1. These findings indicate a significant immunoregulatory role for eosinophils and provide a novel mechanism for the characterization of eosinophil-associated pathologies. This study was supported by the NIH/NHLBI Proteomics Initiative NO1-HV-28184 (AK).

J Biomol Tech. 2009 February; 20(1): 65–66.

SR37-S2 The Effects of Cryptococcus neoformans on Protein Expression in a Human Brain Endothelial Cell Line

Abstract

Cryptococcus neoformans is an invasive opportunistic fungal pathogen that causes life-threatening cryptococcal meningitis in immune-suppressed patients. Untreated cryptococcal meningitis is fatal in normal hosts, and this organism causes high mortality in patients with reduced T-cell-dependent immune function. Unfortunately, treatments are limited because this brain infection remains significantly understudied, and the current antifungal drugs have toxicity and efficacy issues. A number of cellular factors in the brain endothelium are believed to respond and ultimately facilitate the invasion of cryptococcal cells into the CNS; few of these have been identified to date. We have begun to explore the differential protein expression in human brain endothelial cells with or without exposure to C. neoformans. Cells were grown to confluence on standard glass microscope slides, then the cells removed and peptides extracted. Peptides were directly loaded on a Michrom PolySulfoethyl Aspartamide SCX-enrichment microtrap, then sequentially eluted onto a Agilent ZORBAX 300SB C18, reversed phase trap cartridge using (increasingly concentrated) salt injections of ammonium formate. After each salt injection the Agilent C18 trap was switched in-line with a Michrom Magic C18 AQ 100 μm × 150 mm C18 column connected to a Thermo-Finnigan LTQ iontrap mass spectrometer. All MS/MS data were analyzed using X! Tandem, then Scaffold to validate peptide and protein identifications. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides. Our initial analysis identified approximately 500 brain endothelial cell proteins, with approximately 10% of these proteins either up-regulated or down-regulated in response to cryptococcal cells. The proteins identified belong to several different functional groups including cytoskeletal/mobility, immune system, cell signaling, mitochondria-related and oxidative defense. These findings suggest that the interaction between the brain endothelium and cryptotoccal cells is a dynamic process.

J Biomol Tech. 2009 February; 20(1): 66.

SR38-S2 Comparison of 2-Dimensioal Protein Gels from Wild Type and Mutant Yeast Strains as Analyzed by Two Different Instrument Systems

Abstract

Proteomic analysis of biological samples is increasing in its importance for various research applications. Part and parcel of this increase in interest is an influx of commercial formats available for utilizing this technology. Because cost and application are generally limiting factors when choosing among the various instrumentation available, one challenge for a core facility is then to choose among these available formats one which can fulfill the needs of both client and facility. To this end a robust comparative analysis of competing platforms would be beneficial. Our experimental models were a tRNA methyltransferase (Trm10) deletion strain of Saccharomyces cerevisiae and its otherwise isogenic wild-type parent strain. The trm10Δ strain is hypersensitive to the presence of the anti-tumor agent 5-fluorouracil (5-FU), but the biological basis for this hypersensitivity is not known, and identification of proteomic changes that result from growth in the presence of 5-FU could provide important clues to the mechanism of 5-FU action in cells. Following overnight treatment with a sub-lethal dose of 5-FU, total soluble protein from both strains was isolated and a portion was labeled using the GE Healthcare DIGE labeling system and subjected to separation by 2D PAGE. The resulting gels were (1) subsequently visualized on the Bio Rad Versa Doc 4000MP Imaging platform and analyzed with PDQuest and (2) imaged with the GE Health-care Typhoon system and analyzed with DeCyder for comparison of the platforms. The same protein isolates were also separated by 2D PAGE and stained with SYPRO Ruby protein stain and subsequently visualized on both imaging platforms. Images were analyzed using the GE Healthcare DeCyder and Bio Rad PD Quest 8.0 analysis software packages as a further comparison group.

J Biomol Tech. 2009 February; 20(1): 66.

SR39-S2 Dual Mass Spectrometry Approach for the Detection and Quantitation of Active Ricin in Foods

Abstract

The toxin ricin has gained notoriety due to wide availability and potential use as a bioterror agent. Ricin toxin comprises 1–5% of castor beans by weight and is relatively easy to extract. Because of this, there is concern over deliberate contamination of the food supply. Ricin poisoning by ingestion is estimated at 20 μg/kg in humans. Ricin is a 64-kDa ribosome-inactivating protein that causes cell death by blocking protein translation. Several methods exist for detecting ricin, including cell death assay, cell-free translation, PCR, ELISA, and mouse bioassay. Though some of these methods are fast and have low detection limits, they may give false positives, detect only genetic material, or require live cell cultures or animal facilities. None combine quantitation of ricin with a test for activity at the sensitivity and selectivity required. The method described here achieves the goals of speed, sensitivity, and selectivity for ricin analysis in food. Ricin was added to food matrices such as water, half-and-half, 2% milk, and apple juice, and extracted from samples using polyclonal antibodies bound to magnetic beads. To test for activity, the beads were mixed with buffer containing a 12-mer ssDNA substrate and allowed to incubate. Depurination of the substrate was confirmed by MALDI-TOFMS in positive linear mode. Bound ricin was then digested using trypsin and absolute quantitation performed by isotope dilution mass spectrometry. Seven isotopically-labeled tryptic peptides from ricin A and B chains were synthesized and mixed with digests of captured ricin. Two of the peptides allow ricin to be distinguished from its agglutinin. Samples were detected using an LTQ Orbitrap XL operating in production-monitoring mode. Using this method, ricin was successfully extracted from each food matrix tested. Activity of recovered ricin was assessed and quantitation achieved with an LOD of 50 fmol/mL in less than 8 hours.

J Biomol Tech. 2009 February; 20(1): 67.

SR41-S2 ITRAQ Proteomics Revealed Functional Differentiation of Guard Cells and Mesophyll Cells

Abstract

Guard cells are highly specialized cells, forming tiny pores called stomata on leaf surface. The opening and closing of stomata control leaf gas exchange and water transpiration. Mesophyll cells are specialized for photosynthesis. Despite of the phenotypic and functional differences between the two types of cells, the full protein components and their functions have not been explored, but are addressed here through a comparative proteomic approach using iTRAQ tagging and 2D LC-MS/MS, we have identified 1458 non-redundant proteins in the guard cells and mesophyll cells of canola leaves. Numerous proteins were found to be differentially expressed between guard cells and mesophyll cells. Proteins involved in energy (respiration), transport, transcription (nucleosome), cell structure, and signaling are preferentially expressed in guard cells. By contrast, proteins involved in photosynthesis, starch synthesis, disease/defense/stress, and other metabolism are preferentially represented in mesophyll cells. This work represents the most extensive proteomic description of canola guard cells and has improved our knowledge of the functional specification of guard cells and mesophyll cells.

J Biomol Tech. 2009 February; 20(1): 67.

V71-S2 Biomarkers for Acute Contusive Spinal Cord Injury in Rats

Abstract

Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult leading to neuronal dysfunction and death; yet, the molecular mechanisms underlying its dysfunction are poorly understood. The lack of biomarkers for monitoring spinal cord injury (SCI) makes accurate diagnosis and evaluation of SCI progression difficult. In this study, a proteomic approach was used to look for biomarkers from rat spinal cord tissue using iTRAQ labeling and 4800 TOFTOF instrument. Several biomarkers involving in energy metabolism, signaling pathways, protein degradation, inflammatory response, stress response, DNA damage, neuronal functions and blood-spinal cord barrier disruption were identified.

J Biomol Tech. 2009 February; 20(1): 67.

V72-S2 Rapid Pathogen Identification by MALI-TOF Mass SPE Spectrometry/Saramis Database in Clinical Microbiological Routine Diagnostics

Abstract

MALDI-TOF MS/SARAMIS is a straightforward, rapid, robust, and, inexpensive method for the routine identification of bacteria and fungi in clinical microbiology laboratories. Automated identification systems are widely used in medium-to-high-throughput clinical microbiology laboratories. However, such systems are relatively slow because they depend on bacterial growth and metabolic activity. Bacterial identification by MALDI-TOF mass spectrometry provides a promising way to accelerate pathogen identification, since it can be performed in a few minutes from small samples. In this study we compared with 1,400 clinical routine samples the performance of MALDI-TOF MS coupled to SARAMIS (Spectral ARchiving And Microbial Identification System, AnagnosTec, Germany) with established methods (VITEK2/API, BioMérieux) in the clinical microbiology routine diagnostics.

J Biomol Tech. 2009 February; 20(1): 67–68.

V73-S2 MALDI Imaging: Interpretation of Gastric Cancer MALDI Images by Hierarchical Clustering

Abstract

As MALDI imaging draws more attention in the clinical research community, mass spectrometrists are increasingly requested to perform these analyses. The resulting datasets can only be interpreted in the histological context by a pathologist. In a collaboration, the mass spectrometrist is asked to provide the results in an accessible format that allows a quick and intuitive analysis by the pathologist.

A quick way for analyzing MALDI imaging data based on similarity of spectra rather than on individual mass signals is highly desirable. Here, we present hierarchical clustering as a tool to interpret MALDI imaging results and compare them to the histology in an interactive way. Tissue sections (human gastric cancer and mouse organs) were cut on a cryostat and thaw mounted on conductive, ITO-coated glass slides. The MALDI matrix was applied with an imagePrep station. The mass spectra were subjected to hierarchical clustering. Images were reconstructed by selecting nodes in the dendrogram and assigning a color to the respective mass spectra (pixels). Hierarchical clustering is a technique that sorts the mass spectra in a dendrogram according to overall similarity. We found it useful to select individual tree nodes in the full dendrogram and to highlight the respective spectra on the image. This allowed a semi-supervised interactive annotation of the datasets. The results were generally in good agreement with the histology and allowed in some cases even the assignment of histologi-cal structures invisible in individual ion images. Compared to the analysis of MALDI imaging results in a tedious and time-consuming peak-by-peak fashion, the hierarchical clustering allowed a complete annotation of the images in a few minutes, which suits perfectly with the limited time a clinical researcher can spend on this type of work.

J Biomol Tech. 2009 February; 20(1): 68.

SR42-S2 Automating Commercial Mass Spectrometry Search Engines

Abstract

Commercial database search engines for tandem mass spectrometric data offer “black box” capabilities but often ship with additional features that can simplify result preparation for high-throughput core facilities. We demonstrate round-trip sample submission to a Sorcerer PE V3.4 appliance (Sage-N Research, Inc.) that submits and retrieves search results from a Mascot server (version 2.2, Matrix Science), automatically merges results into a consolidated Scaffold result file ready for the user. We installed XCalibur and ReAdW.exe on Sorcerer’s preinstalled CrossOver virtual machine per the Sorcerer installation instructions. This allows direct submission of Thermo .RAW files without prior mzXML conversion. A dedicated user account was created on our Mascot server, and the same databases loaded on each server. Using Sorcerer’s installed TPP software (version 3.4) we generated MGF files from resulting mzXML files using MzXML2Search and submit these files to our Mascot search engine using X11 tunneling to launch an internet browser from the Sorcerer server. Resulting .DAT files were downloaded from Mascot to the ”original” folder generated on Sorcerer for the initial search, and ScaffoldBatch2 was invoked to generate a Scaffold result file incorporating both Sorcerer/SEQUEST and Mascot results. The workflow described above has also been automated using an in-house developed Python program. The Python program monitors all the searches submitted to the Sorcerer, converts mzXML to mgf and submits those searches to Mascot server using the Mascot command-line interface, retrieves the results from the Mascot server to the Sorcerer server using SFTP, and generates the merged Scaffold files. This process is fully automated and can be performed without human supervision. The Python scripts and other documentation will be shared upon request and involve no modification to proprietary vendor code. Supported in part by a grant from The Searle Funds at the Chicago Community Trust to the Chicago Biomedical Consortium.

J Biomol Tech. 2009 February; 20(1): 68.

SR43-S2 Database Analysis of Electron Transfer Dissociation Mass Spectrometry Data

Abstract

Electron Transfer Dissociation (ETD) is a fragmentation mechanism that has recently become widely available in commercial mass spectrometers. The radical-based fragmentation mechanism produces different fragment ion types to the more ubiquitous collision-induced dissociation (CID) fragmentation. Initial reports talked of production of ‘picket-fence’ c and z ion series, but the reality is more complicated, with b and y ions sometimes observed (especially if supplemental activation is used), formation of z+1 and c-1 ions and even amino acid side-chain cleavages. In this poster we will present our experiences analyzing ETD acquired in an LTQ and an Orbitrap by database searching using Protein Prospector, discussing ion types observed and problems with mass accuracy of peaklists. This work is funded by NIH NCRR grant RR001614 and the Vincent J. Coates Foundation.

J Biomol Tech. 2009 February; 20(1): 68–69.

SR44-S2 The PRIDE Database: A Hub for Proteomics Data and Associated Tools

Abstract

One of the early problems encountered by proteomics when it evolved into a high-throughput discovery platform, was the management and dissemination of the accumulated data. Over the past few years, several different data repositories have sprung up to alleviate this need. We here present one of these, the Proteomics Identifications Database (PRIDE), which has been one of the key repositories in the community since its inception in 2005. Since then, PRIDE has evolved with the field in terms of standards compliance, data contents, and available tools. PRIDE is one of the few proteomics repositories today that readily supports secure peer reviewing of submitted data prior to publication, and submission to PRIDE of supporting data for manuscripts is therefore increasingly recommended by high-ranking journals in the field. PRIDE also puts heavy emphasis on sufficient metadata annotation, to ensure the correct interpretation of submitted data. To this end, the widely used Ontology Lookup Service (OLS) was developed within the team. At the same time, PRIDE has greatly expanded the ways in which users can browse and query data, for instance using the powerful PRIDE BioMart and the immensely popular Protein Identifier Cross Referencing (PICR) service. The latter is unique in that it can translate protein identifiers across namespaces as well as across time, seamlessly updating older accession numbers to their newer equivalents. Submitting data to PRIDE also has never been easier, with the freely available PRIDE Converter wizard which efficiently creates fully annotated PRIDE XML from a variety of input formats. Finally, PRIDE also plays an important role in ProteomExchange, the large-scale collaboration to exchange proteomics data between different repositories. The PRIDE database has thus become a real hub for proteomics data and associated tools, and is set to evolve further alongside the field of proteomics over the next years.

J Biomol Tech. 2009 February; 20(1): 69.

SR45-S2 IDPicker 2.0: Improved Protein Assembly with High Discrimination Peptide Identification Filtering

Abstract

Tandem mass spectrometry-based shotgun proteomics has become a widespread technology for analyzing complex protein mixtures. A number of database searching algorithms have been developed to assign peptide sequences to tandem mass spectra. Assembling the peptide identifications to proteins, however, is a challenging issue because many peptides are shared by multiple proteins. IDPicker is an open-source protein assembly tool that derives a minimum protein list from peptide identifications filtered to a specified False Discovery Rate. Here we update IDPicker to increase confident peptide identifications by combining multiple scores produced by database search tools. By segregating peptide identifications on both charge state and the number of tryptic termini in validation, IDPicker yields more confident identifications for protein assembly. The new version achieves higher accuracy by requiring additional novel peptides in the parsimony process, especially when searching against multiple-species databases. IDPicker has been tuned for incorporation in many identification workflows by the addition of a graphical user interface and the ability to read identifications from the pepXML format. These advances position IDPicker for high peptide discrimination, reliable protein assembly, and highly scalable proteomics studies.

J Biomol Tech. 2009 February; 20(1): 69.

SR46-S2 TagRecon: Identification of Amino Acid Mutations in Complex Samples Using Peptide MS/MS Sequence Tags

Abstract

Shotgun proteomics is the preferred tool for large-scale characterization of protein modifications present in complex samples. Numerous search algorithms were developed to characterize known and unknown protein modifications using tandem MS/MS. Peptide sequence tag based search algorithms (like InsPect) can efficiently identify peptides with unanticipated modifications. However, the efficacy of sequence tagging for identifying unanticipated amino acid mutations has not been demonstrated. We developed TagRecon, a novel sequence tag based search engine, to identify amino acid mutations present in complex LC/MS/MS mixtures. TagRecon rapidly scans a database for candidate peptides using short sequence tags derived from a MS/MS spectrum. Candidates are matched to spectra by comparing prefix and suffix masses on either side of a matching tag while allowing for one mass mismatch to occur. Mass mismatches are interpreted as either amino acid mutations or modifications. Finally, probabilistic scores for candidate matches are computed using matched fragment ion intensities and their m/z fidelities. Proteins are inferred from peptide identifications using the IDPicker protein assembly tool. A simulated mutation dataset was generated by analyzing the defined Sigma UPS protein mixture using an LTQ, and introducing random mutations in their corresponding FASTA sequence database. TagRecon was used to match the experimental peptides to the database peptides containing simulated mutations. A total of 93 peptides containing simulated amino acid mutations were identified. 85% of identified simulated mutations were localized to correct residues (accuracy), 4% were incorrectly localized, 4% were chemical modifications mis-interpreted as simulated mutations (at the same mass), and the rest (7%) were false positives. TagRecon reads spectra and outputs peptide identifications using standard formats (mzML and pepXML). It also takes advantage of multi-core CPUs and multi-node clusters. These advances, along with its accuracy, make TagRecon an ideal tool for large-scale detection of amino acid mutations present in complex samples.

J Biomol Tech. 2009 February; 20(1): 69–70.

SR47-S2 A Free Web-Based Database Search Engine for Tandem Mass Spectrometric Data of Proteins and Peptides

Abstract

In the report, we describe a free Web-based database search engine for tandem mass spectrometric data of proteins and peptides. The Web-based search engine consists of two main components (1) MassMatrix database search program (http://www.massmatrix.net), and (2) a Web interface for MassMatrix. MassMatrix is a database search software package for tandem mass spectrometric data. It has additional capabilities that set it apart from other database search engines. It is capable of direct searching of tandem mass spectrometric data of proteins and peptides with intact disulfide bonds or chemical cross-links. The algorithm is also capable of searching through hierarchical MSn (n = 3) spectra (useful in phosphopeptide analysis) where higher confidence in peptide ID can be achieved over MS2 alone. A robust linear regression based algorithm has also been included in MassMatrix to perform automated evaluation of peptide identifications by use of LC retention times to improve the sensitivities and specificities. New features of the automated data analysis for the quantitation of proteins and peptides by use of isobaric MS/MS tag (iTRAQ and Tandem Mass Tag) and N15 labeling have been added to MassMatrix. The Web interface used to access Mass-Matrix is also a generic Web interface for other database search engines. It can be adapted for any database search engine with minor modifications and be run on a PC or a cluster under the Windows or Linux operating systems. The Web interface allows users to configure the database search engine, perform searches, organize and view their results. All settings, configurations, and search results on the Web server are user specific and only accessible to privileged users. Both MassMatrix search program and the Web interface are free and accessible at http://searcher.rrc.uic.edu running on a six-node cluster.

J Biomol Tech. 2009 February; 20(1): 70.

SR48-S2 Integration of Chromatography, LC-MS/MS Data Acquisition, and Peptide Identification Performance Metrics into a Proteomics Software Pipeline

Abstract

Analytical variability is an often overlooked reality for most proteomics workflows. Failure to identify and measure it can undermine biomarker discovery efforts. In this report, we describe a panel of performance metrics targeting six specific areas of LC-MS/MS proteomics platforms. These categories include liquid chromatography, ion source, dynamic sampling, MS, MS/MS and peptide identification. Variability in each these categories is dissected with specific and quantitative metrics which can be used to evaluate technical repeatability (within a series) and reproducibility (between series). These metrics have been implemented in a freely available software pipeline. The pipeline directly processes Thermo Fisher .raw files (from LTQ and LTQ-hybrid mass spectrometers) and includes: (1) a converter/feature-finding algorithm for extracting peak lists, and approximating peak widths and peak heights, (2) a peptide identification engine (SpectraST or OMSSA), (3) a program that calculates all of the metrics using the output files, and (4) a program generating summary statistics reported in a text file. The programs are implemented in C or C++ and the pipeline script is written in Perl. An example application using increasing loading amounts of a yeast reference material will be presented. This software represents a new tool for assessing technical variability and optimizing performance in shotgun proteomics.

J Biomol Tech. 2009 February; 20(1): 70.

V74-S2 Web-Based Solutions for Scientific Associations, Core Labs and Shared Research Resource Laboratories

Abstract

We have created specific Web-based technology for scientific associations and institutions, core labs and shared research resource laboratories. The Web-based technology provides these institutions the ability to manage multiple informational Web sites through one centralized content management (CMS) system. This centralized CMS solution saves administration time, and provides efficiencies in managing different Web sites for one organization. The system administrator can assign any user the ability to edit any set of pages across different Web sites. The granular permissions structure is group based to simplify the routine changing of staff access over time. Page viewing permissions can also be set which restricts the content to a defined set of users.

An online grant application and reviewing module provides the ability to assign grant applications to reviewers, who can then securely review these applications online. The administrator(s) can then electronically approve or reject the grants, and notify the submitters of the decision. The information collected by the grant application can be easily modified as needed. The system supports budgets in a spread sheet format, and all data can be exported for offline analysis. Research and working group functionality allow a collaborative method for posting content and documents to any site. This module is powered by the same users’ permission module as the rest of the technology. Web-based technology developed specifically for the needs of scientific associations, core labs and shared research resource laboratories, can streamline the management of their online presence—saving time and money.

J Biomol Tech. 2009 February; 20(1): 70–71.

V75-S2 Identification of Mosquito Salivary Gland Proteins and Determination of Parasite Infection

Abstract

Mosquito salivary glands are organs specialized in the production of a mixture of molecules that facilitate blood feeding by the lubrication of mouthparts and the inhibition of homeostasis. This is relevant for malaria research since the Plasmodium sporozoites invade salivary glands and are injected with the saliva into vertebrate hosts during blood feeding. To get insights into the proteomics of mosquito salivary glands, the organ was isolated from mosquitos and the proteins separated by 1D-PAGE after cell lysis. The gels were silver stained, bands were cut from the gel and subsequently digested using trypsin. The bands were analyzed by nanoLC-ESI-QTOF-MS/MS on a Bruker micrOTOF-Q II system and all the data transferred into the ProteinScape database system for further data analysis. As expected, in the course of this project, a number of mosquito proteins specific for salivary glands could be identified yet surprisingly not only mosquito-originating proteins were identified but also three proteins originating from the nematode parasite Brugia malayi, which was not expected in the sample. This parasite causes the disease Elephantiasis and the combination of 1D-PAGE and nLC-MSMS analysis of the salivary glands allowed the identification of the parasite infection in the mosquitos. Since no P. falciparum–related proteins were identified, it can be regarded as a confident identification of the parasite infection, accidential identification of three different proteins from a known mosquito parasite can be regarded as very unlikely. ProteinScape performs handling and processing of the data from multiple LC-MS/MS datasets in a fast, convenient and automated manner and allows for automated decoy validation of the results. This removes a major bottleneck in complex proteomics experimental setups with multiple separations, multiple types of MS analyses and the use of multiple search engines.

J Biomol Tech. 2009 February; 20(1): 71.

V76-S2 A Novel Approach to Extract and Share Biological Knowledge from Proteomics Data

Abstract

Proteomics today generates vast amounts of peptide and protein identification data with high accuracy. A growing issue in proteomics is data interpretation, particularly analyzes of protein and peptide identified made by automated database search engines, the subsequent extraction of biologically meaningful information from mass spectrometry experiments and sharing this results with co-workers and collaborators. 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 and enables the sharing of the results. 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 and shared with collaborators. Here we presented the express bioinformatic analysis and comparison of large scale proteomics datasets derived from PRIDE database including an organ-specific proteome map for Arabidopsis thaliana and HUPO projects. Protein identifications were clustered using ProteinCenter algorithms based either on indistinguishable proteins or sequence homology. The results of ProteinCenter data processing will be presented including statistical analysis of over- and under-represented features like gene ontology categories, PFAM annotated proteins, Signal peptide proteins, TM annotated proteins, Enzymes, involvment in KEGG pathways and others.

J Biomol Tech. 2009 February; 20(1): 71.

V77-S2 A New Integrated Bioinformatics Platform for Quantitative Proteomics Studies

Abstract

Comprehensive quantification of changes in protein abundances is one of the most important but also among the most challenging tasks in proteomics. Different kinds of separation techniques, of mass spectrometer types, of identification strategies, and of quantification strategies (e.g., dyes, chemical labeling, label free) do all have their particular strength and weaknesses in terms of protein identification rate as well as quantification accuracy and precision. Therefore, a combination of these techniques is necessary to approach a comprehensive and validated description of biological systems. Due to the high complexity of these workflows and vast amounts of data they produce, a bio-informatics platform is required which allows for efficient data acquisition, analysis, interpretation, validation as well as data reduction and linked information storage. We are developing such a platform based on a central database which allows for sophisticated data warehousing and data mining strategies. The benefits of this system is presented in a study using a cell culture model: Lung carcinoma cells treated and not treated with TGF-beta are analyzed with two distinct technologies: First, label free MS quantification using a high resolution LC-ESI-Q TOF system and a list of regulated targets for protein identification is applied. Second, we investigate the biological system after ICPL labeling for MS quantification, SDS-PAGE separation, and protein identification with an high capacity ion trap mass spectrometer operating in AutoMS(n) mode. Database queries are used to compare the results of both workflows in regard of protein identification and quantification. Combining quantification data from several proteomics work-flows based on an integrated hard- and software platform is applied for an in depth study of changes in carcinoma cells.

J Biomol Tech. 2009 February; 20(1): 71–72.

V78-S2 Generation, Validation and Publication of Proteomics Results Following Publications Guidelines

Abstract

Several initiatives have emerged during the last 2 years that try to establish standards to the Proteomics research community. Results are, e.g., the HUPO PSI data formats (such as mzML) or minimal information requirements (such as MIAPE) and publication guidelines of Proteomics journals such as MCP, Proteomics and others. In addition, initiatives provided first platforms on the Web to accept data along with publication of proteomic data, such as PRIDE. We analyzed the effect of TGF beta on a human lung carcinoma cells in a SILE quantitation experiment. The lysates of treated and untreated cells were labeled using ICPL and separated by means of SDS PAGE. Bands were excised and proteins were digested. The extracted peptides were submitted to RP LC-MS/MS runs. Following separate protein database searches the identification results of all gel bands have been integrated into an overall protein list by the ProteinExtractor algorithm. Following a Decoy validation and quantitative analysis, our bioinformatics platform collected the relevant results and exported them into dedicated report files. These contained the relevant information in the required formats to submit it to journals along with the publication draft.

J Biomol Tech. 2009 February; 20(1): 72.

V79-S2 From: Workflow for Maximizing Protein Identifications on the LTQ Orbitrap XL ETD

Abstract

Identifying a maximum number of proteins with the largest sequence coverage in a complex sample with a minimum of effort has always been a challenge. Hereby we describe a method that combines an automated intelligent acquisition method on an LTQ Orbitrap XL using different dissociation techniques with a data processing workflow that uses multiple search engines to increase the number of identified proteins to a maximum. The acquisition method consists of a Decision Tree Data Dependent method, where activation type, CID or ETD, is determined for each peptide based on m/z and charge state to maximize the chance of being identified. The sample is injected multiple times. After each injection all precursors that have been selected for MS2 are added to the exclusion list of the next run automatically. The consecutive runs are searched in Proteome Discoverer with four different algorithms, Sequest, ZCore, Mascot and Peaks DeNovo and summarized.

J Biomol Tech. 2009 February; 20(1): 73.

RG1-S1 The DNA Sequencing Research Group general survey, 2009: Second Generation Sequencing Instruments and Services in Core Facilities

Abstract

The ABRF DNA Sequencing Research Group (DSRG) has conducted a general survey to collect data on the current state of second generation sequencing instrumentation (often termed “massively-parallel” or “next generation” sequencers) and services offered by core facilities. The DSRG has monitored trends in sequencing platforms in core facilities by conducting surveys periodically. However, this survey was the first to focus on second generation sequencers since their introduction. The information gathered this year provided data about the widespread availability of this equipment and these services in core facilities. The future acquisition and expectations for such instruments were also assessed. For comparison, the survey gathered information on Sanger (first generation) sequencing operations to determine how these technologies are affected by the second generation technologies. The importance of this survey lies in the fact that it serves as an initial “snapshot” of the status of second generation sequencing services in core facilities while they are in their infancy, and therefore is a baseline for surveys in years to come.

J Biomol Tech. 2009 February; 20(1): 73.

RG2-S1 Nucleic Acid Research Group 2008–2009 Study: A Comparison of Different Priming Strategies for cDNA Synthesis by Reverse Transcriptase, as Evaluated by Real-Time RT-qPCR

Abstract

Real-time reverse transcriptase quantitative PCR (RT-qPCR) is widely used for measuring transcription levels. Assay-specific primers, although ideal for cDNA synthesis, are not always practical. Priming strategy and reverse transcriptase enzyme affect the sensitivity and variability of RT-qPCR and microarray results. The Nucleic Acid Research Group (NARG) designed a study to determine the optimal priming strategy for RT-qPCR. The NARG 2008–09 study was an extension of the 2007–08 study in which we evaluated the effect of reverse transcription priming strategies on RT-qPCR results. The previous study suggested a relationship between the assay sensitivity using cDNA generated with oligo-dT primers and qPCR assay placement relative to the 3′ end of the transcript. This year’s study was designed specifically to compare oligo-dT and random priming strategies as the assay target site varied. Because the previous study identified random hexamers or nonam-ers as most efficient of those tested, this years study was designed specifically to compare oligo-dT, random 6-mers and 9-mers or gene specific primers and combinations. Four reverse transcriptases; Superscript II, Superscript III, Transcriptor and MultiScribe, were employed to determine the effect of enzyme. In addition, the qPCR assays looked at three genes of varying abundance, β-actin (high copy), β-glucuronidase (medium copy) and TATA binding protein (low copy) as well as varying distance from the 3-prime end for each transcript.

J Biomol Tech. 2009 February; 20(1): 73–74.

RG3-S1 Edman Sequencing Research Group (ESRG) Study 2009: Comparison of Edman and Mass Spectrometry Techniques for N-terminal Sequencing

Abstract

For decades, automated Edman sequencing has been the method of choice for determining the N-terminal amino acid sequence of proteins. However, the major but by no means only limitation of this technique is its inability to obtain amino acid sequence from N-terminally blocked proteins. Mass spectrometric techniques for protein sequence analysis do not suffer from this limitation though unequivocal determination of protein N-termini on a routine basis has been elusive. The advantages of mass spectrometric techniques have in recent years driven investigators to look beyond Edman chemistry to find alternative technologies to obtain N-terminal sequence. Several mass spectrometric methodologies have been published, primarily for proteomics analyses, which may be quicker, less costly and more sensitive than Edman sequencing. Because such techniques involve a range of biochemical and instrumental methodologies having different advantages and limitations the ESRG has created a study to ascertain how reliably they can produce N-terminal amino acid sequence information and to compare those results to those obtained by automated Edman sequencing.

The ESRG 2009 study was designed to allow the participants freedom to use their analytical technique of choice to obtain as much N-terminal amino acid sequence information as possible from two test proteins. Approximately one nanomole of each sample was provided so laboratories may attempt a variety of techniques with the goal of obtaining each protein’s N-terminal sequence. Results of these analyses as well as a comparison of methodology, instrumentation and specific protocols used will be presented so that core facilities can gauge their competence and expectations for determining N-terminal amino acid sequences.

J Biomol Tech. 2009 February; 20(1): 74.

RG4-S1 PRG 2009 Study: Relative Protein Quantification in a Clinical Matrix

Abstract

The Proteomics Research Group (PRG) of the ABRF developed the 2009 study to assess approaches that individual laboratories would use to determine the relative abundance of target proteins in a complex mixture. An increasingly common request for proteomics laboratories is the detection of a specific target protein of interest in a complex mixture. Likewise, most of these requests are also interested in knowing the abundance of the target protein relative to that in a control sample. While this type of analysis has traditionally been addressed using Western blots or other immunoaffinity assay, recent advances in targeted mass spectrometry-based analyses are beginning to be reported in the literature as an alternative.

For this year’s study, four different proteins were spiked into a plasma background matrix at three different levels. Two of these proteins are commonly measured plasma protein biomarkers, and the remaining two had identical primary structure and differed by only a single phosphorylation site. The participants were shipped six samples in total (three samples in blinded duplicate) and asked to report the relative abundances of the four target proteins in the six samples. Results from analysis of the samples and survey responses will be used to assess the different approaches that are used by the proteomics community to determine the relative abundance of a target protein of interest. For this year’s study, four different proteins were spiked into a plasma background matrix at three different levels. Two of these proteins are commonly measured plasma protein biomarkers, and the remaining two had identical primary structure and differed by only a single phosphorylation site. The participants were shipped six samples in total (three samples in blinded duplicate) and asked to report the relative abundances of the four target proteins in the six samples. Results from analysis of the samples and survey responses will be used to assess the different approaches that are used by the proteomics community to determine the relative abundance of a target protein of interest.

J Biomol Tech. 2009 February; 20(1): 74.

RG5-S1 sPRG2009 Study: Development of a Quantitative Proteomics Standards

Abstract

The Proteomics Standards Research Group (sPRG) initiated a study in 2007 that focused on development of a mixture of standard proteins that contained appropriate stable isotope labeled (SIL) peptides and could be used as a model for quantitative plasma proteomics. A set of 350 human plasma proteins was evaluated extensively, with the goal of selecting 50 proteins that would be distributed over five orders of magnitude in concentration. After lengthy consideration of the project, it was decided that there would be too many challenges associated with analysis of this type of sample. Efforts have now focused on development of a simplified standard that is based on human plasma proteins and would be suitable for use in assessing a laboratory’s capabilities for absolute quantitative analysis. Drawing from the information gained previously from evaluation of the 350 human plasma proteins, 10 candidate proteins were selected for further consideration. As a first step, the sPRG members digested and analyzed each of the individual proteins. From those results, a list of prospective peptides was generated and corresponding unlabeled and SIL peptides were synthesized. For the next stage, the sPRG analyzed individual samples containing the synthetic peptides (labeled and unlabeled) for each protein. Subsequently, a digest of a mixture of the proteins was analyzed in the presence of the SIL peptides, and the relative intensities of each unlabeled/labeled peptide pair were assessed. It was concluded from these experiments that better standardization was needed for the proteins and SIL peptides before a study sample could be prepared. This led to a timeline of presenting analysis of a newly-formulated study sample by sPRG members at the ABRF conference and having results from participating laboratories available in time for presentation at ASMS 2009.

J Biomol Tech. 2009 February; 20(1): 75.

RG6-S1 iPRG2009 Study: Testing for Qualitative Differences Between Samples in MS/MS Proteomics Datasets

Abstract

Determining significant differences between mass spectrometry datasets from biological samples is one of the major challenges for proteome informatics. Accurate and reproducible protein quantitation in complex samples in the face of biological and technical variability has long been a desired goal for proteomics. The ability to apply qualitative difference testing is a first step towards that goal, and is routinely used in tasks such as biomarker discovery. In this work the Proteome Informatics Research Group (iPRG) of the ABRF presents the results of a collaborative study focusing on the determination of significantly different proteins between two complex samples. In this study, datasets representing five technical replicates of each sample were provided to volunteer participants and their ability to evaluate reproducible differences was tested. A survey was used to determine the relative merits of spectrum counting versus MS intensity-based differentiation, whether sophisticated statistical methods are necessary, and if computer software must be augmented by scientific expertise and intuition. Results and survey responses were used to assess the present status of the field and to provide a benchmark for qualitative difference testing on a realistically complex dataset.

J Biomol Tech. 2009 February; 20(1): 76.

CF2-M Cornell University Life Sciences Core Laboratories Center

Abstract

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.

J Biomol Tech. 2009 February; 20(1): 76.

CF3-M Information technology Services Facility of the Cornell University Life Sciences Core Laboratories Center

Abstract

The Information Technology Services Facility of the Cornell University Life Sciences Core Laboratories Center (CLC) provides desktop and network support, software license management, and LIMS maintenance and development to our core facilities as well as a diverse array of Cornell University life sciences investigators and academic units.

J Biomol Tech. 2009 February; 20(1): 76.

CF4-M New in the CGRB Core Laboratories

Abstract

The Center for Genome Research and Biocomputing Core Lab at Oregon State University provides services for fee in genomic technologies (DNA sequencing, DNA fragment analysis (genotyping)) and in functional genomic technologies (microarray). Sequencing is provided both for traditional Sanger sequencing on an AB 3730 and Ultra highthroughput sequencing on the Illumina Genome Analyzer. This year we added the Paired End module and upgraded to the GAII. DNA fragment analysis is performed on the AB 3100. Our microarray services include Affymetrix and NimbleGen platforms. Sample labeling, hybridization and scanning are offered. NimbleGen capabilities were added this year. The Agilent BioAnalyzer is a service often tied to the microarray services. We also have a BioRobotics Microgrid for spotting custom microarrays. Our biocomputing infrastructure has grown to include over 110 machines and over 450 processors. We maintain over 100 TB of shared file storage, gigabit-ethernet network switches with a 10-gig backbone, an 800/1600GB tape backup system and standardized Linux cluster node software and operating systems. Policies have been developed to manage the massive data output of the Illumina Genome Analyzer to allow researchers adequate time to analyze their data and to allow Center staff to rotate new data into the infrastructure and older data off. The CGRB also maintains multi-user instruments available to researchers. These include a Zeiss LSM510Meta microscope, an AB7500 qPCR, a Storm 820 Phosphoimager, a Nanodrop, a Genetix Q-Pix colony picker, an Axon Genepix 4200A microarray scanner, and a fluorescent plate reader.

J Biomol Tech. 2009 February; 20(1): 76–77.

CF5-M Idaho State University Molecular Research Core Facility

Abstract

The Idaho State University Molecular Research Core Facility (ISU MRCF) provides shared research instrumentation and services to ISU faculty, staff, students, and external researchers. The MRCF was established in 1994 through a National Institutes of Health/EPSCoR Institutional Development Award. Since 1994 the MRCF has undergone two expansions and currently comprises nearly 1200 square feet in the Gale Life Sciences Building. Facility staff maintain and provide training for 30 scientific instruments. Our current space is in contrast to 173 square feet and six instruments in 1995. The MRCF operates under both the Office of Research and the ISU Biomedical Research Institute and receives support from an Advisory Committee comprised of the MRCF Director, Facility Director, and faculty members representing the Departments of Biological Sciences and Biomedical and Pharmaceutical Sciences. The facility provides DNA sequencing and fragment analysis services using an Applied Biosystems 3130XL Genetic Analyzer. Researchers submit requests and receive data in a streamlined fashion through our Geospiza Finch server, which allows for after hours order requests and around the clock remote access to data files. Shared instrumentation includes the following: PCR, quantitative-PCR, gel imager, microarray, bioanalyzer, DNA/RNA/Protein spectrophotometer, centrifuges, phosphorimager, liquid scintillation analyzer, microplate reader, liquid handling robot, and fluorescence and deconvolution microscopes. Software designed for downstream data analysis is available on several computer kiosks. Authorized researchers have 24 hour access through a card tracking security system. In addition to the critical research component, the MRCF participates in educating the next generation of scientists by providing tours and demonstrations. For example, in November 2008 over 400 students toured the MRCF and were given demonstrations on a variety of instruments. The MRCF also sponsors workshops and seminars such as the annual ISU Bioinformatics Workshop.

J Biomol Tech. 2009 February; 20(1): 77.

CF6-M The SOLiD at Penn State—Implementation of a New Technology in a Core Facility

Abstract

The SOLiD next generation sequencer performs short read sequencing by ligation using DNA amplified on beads and then deposited on a slide. The investment in the SOLiD instrument, accessories, reagents, and computer systems can reach $600,000 and higher. As we implement the SOLiD technology we have been working on reducing costs to the researcher, establishing an efficient work-flow, and optimizing protocols. In particular we have found that sample quality is important in obtaining sufficient yield during library preparation. Accurate titration of the library immediately prior ePCR was the biggest impediment to getting good and best beads. Therefore, we investigated various quantification methods which included the Agilent Bioanalyzer, Nanodrop spectrophotometer, qPCR by SYBR Green and the Qubit fluorimeter. In order to reduce costs we have also been assessing protocols for incorporating barcoding of libraries which will allow multiplexing up to 16 samples per slide section. Another area of cost reduction is to use less Taq Polymerase in the emulsion PCR and we have included PCR mini procedures as routine sample preparation. To aid in data distribution to researchers as well as facilitate further data analysis, a “pipeline” to the galaxy site set up by Anton Nekrutenko here at PSU has been established. Costs and charges will also be discussed.

J Biomol Tech. 2009 February; 20(1): 77.

CF7-M Searchable Core Facility Database: Building Resource Bridges

Abstract

The Vermont Genetics Network was established in 2001 and is funded by the National Center for Research Resources (NCRR), and is part of a National Institutes of Health (NIH) initiative called IDeA Networks of Biomedical Research Excellence (INBRE) to build biomedical research infrastructure. We identify the need for and develop facilities at the University of Vermont (UVM), our lead institution and promote and encourage access for the scientific community throughout the state. Not all infrastructure needs are required by a broad base of local researchers and in these cases would be beneficial to explore accessing these technologies regionally. To explore what services or technologies that are accessible regionally is a desirable goal of the Vermont Genetics Network. This would avoid duplicating a resource that would not be cost effective at one site or state, but could still be accessed regionally. To explore regional networks, an online core facility searchable database has been developed. The database will only be useful if it is populated. A representative of the Vermont Genetics Network will have computers accessible to all ABRF attendees to view and populate. Researchers will be able to search online by service offerings and location to find a facility regionally that will best meet their needs. The data is also available in an excel readable XML file.

J Biomol Tech. 2009 February; 20(1): 77.

CF8-M The Genomic Technologies Facility at Iowa State University

Abstract

Established in 1999, the Genomic Technologies Facility of the Center for Plant Genomics of the Plant Sciences Institute provides training and fee-based services for biological research at the genomic level. Services include but not limited to the production and analysis of custom spotted microarrays for global mRNA profiling experiments; high-throughput genotyping and quantitative gene expression analysis using Sequenom MassARRAY, Stratagene Mx4000 and Roche LightCycler 480 instruments. The Genomic Technologies Facility not only provides expertise and equipment to investigators and collaborators within Iowa State University, but also offers our services outside. The Genomic Technologies Facility and the Schnable research group have combined microarray technology with laser capture microdissection to generate a series of SAM (shoot apical meristems)-enriched maize B73 cDNA gene chips. About 2800 genetically mapped cDNAs have been included on the maize chips, and the MicroArray Data Interface (MADI) is available online to facilitate researchers in querying and viewing information associated with spots printed on these chips. In addition, the Schnable research group has validated more than 1000 SNPs between the two inbred lines B73 and Mo17, which are available for use in a wide variety of maize genetic experiments in the Genomic Technology Facility.

J Biomol Tech. 2009 February; 20(1): 77–78.

CF9-M CIAN—Cell Imaging and Analysis Network

Abstract

The Cell Imaging and Analysis Network (CIAN) provides services and tools to researchers in the field of cell biology from within or outside the McGill University community. CIAN is composed of six scientific platforms: Cell Imaging (confocal and fluorescence microscopy), Proteomics (2D, DiGE and fluorescent protein analyses), Automation-High throughput screening (Pinning robot and liquid handler), Protein expression and antibody production, Genomics (microarray and real-time PCR), and Data storage/analysis (cluster, server and workstations). Users submit project proposals and can obtain training in any aspect of the facility. Since its opening, CIAN served 150 users from 60 labs with 25 affiliations. CIAN is designed to facilitate training, enhance interactions, and share resources and expertise.

J Biomol Tech. 2009 February; 20(1): 78.

CF10-M The Center for Functional Genomics Core facilities at the University at Albany-SUNY

Abstract

The Center for Functional Genomics (www.cfgbiotech.org) is a fee-for-service basic research laboratory providing services to hundreds of researchers at companies, universities, and government agencies around the world, helping them obtain robust, reliable results quickly and affordably. This state-of the-art facility is comprised of multiple technology based core laboratories that offer services in molecular genetics, proteomics, mass spectrometry, microarrays, transgenics, cell culture, laser capture microdissection and flow cytometry. The microarray services include expression analysis using Affymetrix, Agilent and Nimblegen arrays, Eppendorf DualChips or Custom spotted cDNA arrays; ChIP-Chip, RIP-Chip, genotyping using Affymetrix SNP and Targeted Genotyping techonolgies, and qPCR validation. The molecular genetics core provides services in DNA sequencing, genotyping, gene cloning/vector construction, qPCR and gene/promoter isolation. The transgenics core provides transgenic mice and gene targeting technologies for in vivo gene function analysis and generation of animal models. This is done by either pronuclear injection, or mouse ES cell electroporation and subsequent blastocyst injection. The proteomics and mass spectrometry core offers expertise in 2D gel electrophoresis; mass-spec based quantitative protein profiling, post translational protein modification and metabolite ID and quantitation. The flow cytometry core supports instrumentation for multi-color phenotyping, cell cycle analysis and cell sorting. The co-localization of these core facilities offers a seamless and comprehensive approach to drug discovery and health research.

J Biomol Tech. 2009 February; 20(1): 78.

CF11-M Molecular Resource Facility, UMDNJ-New Jersey Medical School

Abstract

The Molecular Resource Facility of the New Jersey Medical School was established in April 1995 to enhance the resources available to the research community within the medical school. It serves to provide services to the research community and as a source of information on molecular techniques and research strategies involving molecular biology. Our facility now provides services in many areas including DNA sequencing, protein sequencing, peptide synthesis, qPCR and others. Our services are available to any laboratory requiring these services.

J Biomol Tech. 2009 February; 20(1): 78.

CF12-M Proteomics, Biological Mass Spectrometry, and High Throughput Inhibitor Screening at the University of Cincinnati

Abstract

The University of Cincinnati Proteomics laboratory offers a full array of proteomics and biological mass spectrometry capabilities both on a fee-for-service basis and as grant-supported collaborative research projects. During the initial consultation with Proteomics Laboratory personnel, the needs of the investigator are addressed and then the appropriate strategy to solve the problem at hand is presented. Through these initial discussions, the overwhelming majority of problems that often plague core laboratories are circumvented thus setting appropriate expectation for the investigator and maximizing the use of the core resources. The capabilities offered range from simple QA analysis of isolated proteins and peptides, to more complex comparative profiling of proteins from two or more biological conditions, and finally to the development technologies in global phosphorylation changes and drug discovery applications of mass spectrometry for high throughput inhibitor screening. For comparative profiling of cells or tissues, both 2D gel electrophoresis approaches and quantitative mass spectrometry methods are offered. 2D gel electrophoresis can be done using the DIGE method for pair wise comparisons or via silver-stained methods and comprehensive image analysis for multivariate experiments. Protein identification from gel spots with sensitivity in the low femtomole to attomole range is routinely done in the laboratory using both nanoLC-MS/MS and MALDI-TOF/TOF methods. Using the same mass spectrometry techniques, identification and characterization of isolated protein complexes are also available. For MS-based quantitative proteomics, currently the preferred method is via isotope labeling using iTRAQ reagents from Applied Biosystems, but analysis for SILAC-labeled culture samples can also be provided. Furthermore, this presentation highlights our expertise in global mapping of phosphorylation sites from complex mixtures by affinity enrichment (TiO2 or IMAC) followed by ion exchange separation and nanoLC-MS/MS. Finally, an emerging application for mass spectrometry-based enzyme assays and inhibitor screening is presented with an eye toward attracting new customers and collaborators outside of the University of Cincinnati who are interested in screening a target enzyme for new inhibitors.

J Biomol Tech. 2009 February; 20(1): 79.

CF13-M Trudeau Institute Molecular Biology Core Facility

Abstract

The core facility paradigm is constantly changing. The Molecular Biology Core Facility (MBCF) at the Trudeau Institute thrives by providing a variety of services to the investigators at the institute while recovering the majority of its expenses. The MBCF offers an assortment of services that are specifically tailored to the Institute mission and personalizes 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 (FACS) analysis, real-time PCR measurement of gene expression and viral loads, knock-out mouse and Mycoplasma screening, RNA quality analysis by using “Lab on a Chip” technology, DNA haptenation and recombinant protein expression/purification for antibody detection and in vivo vaccine development strategies. 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.

J Biomol Tech. 2009 February; 20(1): 79.

CF14-M From Bench to Bedside: Patient-Oriented Core Programs at Nemours Biomedical Research

Abstract

Nemours is one of 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 has a long-standing commitment 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 that bridge the gap between bench and bedside, as well as in programs and services that utilize this knowledge, to impact medical care and disease prevention.

This poster will describe the different CLIA and CAP certified diagnostic cores available for clinicians to help with diagnostic and care of pediatric patients. We will also present an overview of the cores and shared facilities available at the Center for Pediatric Research, a COBRE-funded research center which was created at the Alfred I duPont Hospital for Children to foster the development of translational research programs covering a broad range of pediatric disorders.

J Biomol Tech. 2009 February; 20(1): 79.

CF15-M Quality Assurance and Quality Control in the Core Genotyping Facility

Abstract

The Core Genotyping Facility’s Quality Assurance and Quality Control Group provides the necessary resources in support of a controlled laboratory environment, resulting in efficient DNA sample handling, robust, reliable and reproducible genotyping, and streamlined data analysis. Members of this group work closely with the staff of the Production Genotyping Team, Dedicated Scientific Operations, and Bioinformatics groups to create Standard Operating Procedures for laboratory and analysis workflows. In addition to SOP development and training, members are also responsible for laboratory automation, equipment maintenance, manufacturing of sample handling and genotyping controls and standards, and new assay validation. Select members of this group also review pre-genotyping sample eligibility and perform post-genotyping quality control analyses, creating a highly informative dataset for genetic epidemiological analyses. Together, this group ensures that the product delivered meets the customer’s requirements for high quality data, and that data is generated following the most efficient and cost-effective laboratory and analytical procedures.

J Biomol Tech. 2009 February; 20(1): 79–80.

CF16-M DNA Sequencing and Genotyping Facility

Abstract

The DNA Sequencing and Genotyping Facility of the Cornell University Life Sciences Core Laboratories Center (CLC) provides an array of shared research resources and services to the university community and to outside investigators. The facility offers a concentration of advanced instrumentation and expertise in their applications. Services include the ABI3730xl platform for Sanger sequencing of plasmid and PCR products. Resources for massively-parallel, “next generation” sequencing include the Roche 454 GS FLX and the Illumina Genome Analyzer II. The Sequenom MassArray is available for SNP genotyping, methylation and gene expression studies. The ABI 7900HT is available for RT-PCR studies. The facility also provides support for SNP genotyping using automated sample processing pipelines for ABI SNPlex, Illumina GoldenGate and Infinium, and Affymetrix SNP and Targeted Genotyping projects. The goal of the facility is to meet the increasing need of investigators for rapid and accurate DNA sequencing and genotyping.

J Biomol Tech. 2009 February; 20(1): 80.

CF17-M Applying Next Generation Sequencing Technologies as Shared Research Resources

Abstract

New DNA sequencing technologies presents an exceptional opportunity for novel and creative applications with the potential for breakthrough discoveries. To support such research efforts, we have implemented the Illumina Solexa Genome Analyzer II and the Roche 454 Genome Sequencer FLX platforms as academic core facility shared research resources. We have established sample handling methods and informatics analysis 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 de novo or reference based genome assembly, detection of genetic variation, transcriptome sequencing, small RNA profiling, and genome-wide measurements of epigenomic 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.

J Biomol Tech. 2009 February; 20(1): 80.

CF18-M Services Offered by the DNA Sequencing and Genomics Core Facility at the University of Utah

Abstract

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. Our sequencing facility 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, we offer mitochondrial genome sequencing, a mutation detection service to identify single nucleotide polymorphisms (SNPs), insertions, and deletions. Researchers can select genes or regions of interest to which 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 facility 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 microsatellite 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. Taqman genotyping assays and higher throughput custom SNP genotyping can know be achieved using the Illumina Bead Express system. To meet the real-time PCR gene expression demands of our customers, two Applied Biosystems 7900HT instruments are available. Additionally, we have two automated fluid handling systems, a Biomek FX and a Velocity 11 VPrep for researchers needing automated fluid handling for their experiments.

J Biomol Tech. 2009 February; 20(1): 80–81.

CF19-M SOLiD Sequencing Enables Studying the Whole Methylome

Abstract

Through the addition of methyl groups to Cytosine (C) at CpG sites (Cytosine and Guanine separated by a phosphate), transcription of the DNA is inhibited. Thus methylation plays an important role in the regulation of gene expression in both the normal and dysfunctional cell. It can be found in numerous cell processes, such as differentiation, chromosome stability, the transcription prevention of repetitive or alien sequences, and cancer development. During bisulphite conversion and subsequent PCR amplification, unmethylated Cs are converted into Thymine (T), while methylated Cs will not be converted. Sequencing of this bisulphite treated DNA, enables the characterization of the methylation status for each CpG site. However only the introduction of next generation sequencing technologies have overcome the limited analysis of CpGs in specific regions, thus providing the opportunity for hypothesis-free study of the entire methylome. Here we present a technique that utilizes the high throughput of the SOLiD™ System with the single base level resolution of bisulphite sequencing. A novel adaptation to the fragment library protocol enabled the DNA library to be treated with bisulphite after the addition of sequencing adapters. The resulting bisulphite converted library was then subjected to emulsion PCR and sequencing using standard sequencing protocols. The conversion of unmethylated C to T during the bisulphite reaction essentially reduces the DNA to a three base genome, typically making mapping of short reads very challenging. However using the SOLiD 2 base encoding system, the three base bisulphite converted genome retains all four colors in color space, thus providing the complexity required for accurate mapping.

J Biomol Tech. 2009 February; 20(1): 81.

CF20-M RWJMS DNA Core Facility Provides Fast and Accurate DNA Sequencing and Genetic Analysis Services

Abstract

The RWJMS DNA Core Facility provides automated DNA sequencing and genotyping services, oligonucleotide synthesis, DNA fragment analysis, SNP analysis, Human cell marker identification services, Taqman low density arrays, quantitative Real-Time PCR services and an ABI Freezer Program. The Facility runs an ABI PRISM® 3100 Genetic Analyzer and a 3130XL Genetic Analyzer, state of the art machines for automated DNA capillary electrophoresis sequencing and genotyping. The Core utilizes an ABI 7900HT Real-Time PCR System that can accommodate any real-time PCR application including Taqman® Assays. This high-throughput system contains robotics enabling multiple 384 or 96 well plates to be loaded automatically. The Core also has an agreement with Integrated DNA Technologies, an innovative leader in DNA synthesis, to provide fast, quality DNA and RNA oligonucleotides at a greatly reduced cost with no shipping charges. In addition, the DNA Core Facility purchases select Applied Biosystems reagents in bulk. We are able to pass this cost-savings onto our users with our ABI Freezer Program. The DNA Core Facility is a shared resource of the Cancer Institute of New Jersey.

J Biomol Tech. 2009 February; 20(1): 81.

CF21-M NCI Core Genotyping Facility: Technology Enables Genome-Wide Association Studies

Abstract

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, bio-statisticians and basic research scientists in the intramural research program, the CGF has developed the capacity to conduct genome-wide association studies (GWAS) and candidate gene approaches to identify the heritable determinants of various forms of cancer. Utilizing technologies by Illumina®, Affymetrix®, Applied BioSystems®, Fluidigm®, and Roche/454®, the CGF has created a laboratory facility capable of high-throughput genotyping and detailed scientific follow-ups studies. 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 added the Roche/454® next-generation sequencing platform to allow deep re-sequencing and follow-up of regions of interest found with the various SNP genotyping techniques. Funded by NCI Contract HHSN261200800001E.

J Biomol Tech. 2009 February; 20(1): 81.

CF22-M Epigenomics Facility

Abstract

The Epigenomics Facility of the Weill Cornell Medical College and the Cornell University Life Sciences Core Laboratories Center (CLC) provides an array of epigenomics research resources and services to the university community and to outside investigators. The facility offers a concentration of advanced instrumentation and expertise in their applications. This is an inter-campus facility, with resources and services located both on the Cornell University campus in Ithaca, NY, and the Weill Cornell Medical College in NYC, NY. DNA methylation profiling and protein-nucleic acid association analysis (ChIP-Chip and ChIP-Seq) resources include the NimbleGen Illumina, Affymetrix, and Agilent microarray platforms, the Sequenom MassArray, the ABI 3730xl, and the Roche 454 GS FLX and Illumina Genome Analyzer II. The goal of the facility is to meet the increasing need of investigators for rapid and accurate epigenomics project design, sample preparation, data generation, and data analysis.

J Biomol Tech. 2009 February; 20(1): 81–82.

CF23-M Core Biology Facility at North Dakota State University

Abstract

The Core Biology Facility (CBF) was established at NDSU in 2003 with NCRR-NIH-COBRE grant funding to provide access to equipment and specialized training in the areas of molecular biology and tissue culture in order to enhance the biomedical research environment at NDSU. The CBF is also instrumental in testing compounds synthesized by NDSU’s Chemistry faculty for potential inhibition of matrix metalloproteinases and histone deacetylase enzymes. To assist clients in performing their experiments, the CBF is equipped with a Gemini EM microplate fluorimeter, a Thermo Multiskan spectrophotometer, an Agilent 2100 Bioanalyzer, two ABI 7500 Fast Real Time PCR machines, two ABI 2720 thermocyclers, an Axon 4000B Genepix microarray scanner, an Accuri C6 Flow Cytometer, a computer workstation with FlowJo 8.7.1 and access to the IPA Ingenuity Database, and a Leica DMIL inverted microscope with digital color camera, in addition to basic molecular biology and tissue culture instrumentation. The CBF also provides investigator or student training and consultations on experimental design and data interpretation.

J Biomol Tech. 2009 February; 20(1): 82.

CF24-M Quantitative PCR Services in SeqWright

Abstract

SeqWright since its inception 14 year ago has grown to become a premier genomics service provider offering its customer access to the latest cutting edge technologies in the genomics service arena. Quantitative PCR(QPCR) services were offered as a natural addition to the SeqWright portfolio of services which included DNA sequencing, complex gene cloning, cell line ID, and genotyping. SeqWright’s QPCR service is run in a state of the art facility by seasoned industrial scientists with many years of experience. With the recent acquisition of the ABI 7900HT to increase capacity, we are now able to handle any scale of project. With a reputation for excellence, SeqWright has recently become a preferred ABI QPCR service provider.

We have established SOP and validation of all the major applications for QPCR and can offer services at both research and regulatory levels. We offer the following QPCR services: copy number determination of DNA or RNA targets, absolute or relative quantitation with a choice of normalizing methods, gene expression assays on over 800,000 genes in 9 species using the pre-designed Taq-Man® assays, microRNA assays and microRNA profiling for a broad range of species, microarray study validation as a companion service to its world leading microarray service, post-PCR SNP genotyping and plus/minus assays.

Benefits of SeqWright’s QPCR service include: dedicated Ph.D. project manager providing careful consideration of assay design, template preparation, and analytical methods for each individual QPCR project. Capacity and process automation required to handle any scale of project. Support for both research and regulatory levels of compliance.

J Biomol Tech. 2009 February; 20(1): 82.

CF26-M Danforth Center: Proteomics & Mass Spectrometry Core Facility

Abstract

The Proteomics and Mass Spectrometry Facility at the Donald Danforth Plant Science Center (http://www.danforthcenter.org/pmsf/) 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 on-line 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 postranslational modification analysis. We currently have 4000 QTRAP methods for plant hormone quantitation, folate analysis, and ionic compound profiling. Additionally, we have a Waters UPLC system set-up to perform both free and hydrolyzed amino acid analyses.

J Biomol Tech. 2009 February; 20(1): 82–83.

CF27-M University of Massachusetts Worcester Foundation Campus Laboratory for Proteomic Mass Spectrometry, Shrewsbury, MA

Abstract

The laboratory was originally established in 1989 with a grant from the W.M. Keck Foundation given to the former Worcester Foundation for Biomedical Research. The laboratory’s technology focus has evolved over the last decade from a largely micro chemical approach to characterizing proteins (Automated Edman Degradation) to a mass spectrometry based approach. Our services are offered to a large clientele from both academic and corporate entities in the US and abroad. The laboratory offers both ESI and MALDI based ionization mass spectrometry services however we have focused on the development of very high sensitivity gel based analyses using advanced state-of-the-art MALDI TOF mass spectrometry. The two MALDI TOF instruments from Shimadzu Biotech (Axima QIT, and Axima TOF2) provide both accurate MS and true tandem (MS/MS) capabilities at sub femptomole levels for peptides. This poster will give some examples of the kinds of problems addressed utilizing the various instrumentation systems. Such examples will include protein identification, de novo sequencing, and the localization of both chemical and post-translational modifications.

J Biomol Tech. 2009 February; 20(1): 83.

CF28-M FC2: Analytical Instrumentation—Biological Mass Spectrometry

Abstract

The Analytical Instrumentation Facility Core is a biological mass spectrometry facility that performs service work and collaborative research for the biology, pharmacy and biomedical researchers at the University of Texas at Austin and members of the Center for Research in Environmental Disease at M.D. Anderson Cancer Center and the UT School of Public Health in Houston. The core has MALDI-TOF, MALDI-TOF/TOF, ESI-ion trap, ESI-triple quadrupole-linear ion trap, and GC-quadrupole mass spectrometers. In addition, there are HPLC systems from analytical to nanoflow range and an HPLC system coupled to an electrochemical (EC) detector. Our focus is on biological molecules ranging from metabolites, drugs, and nutrients to protein analysis. We conduct quantitative analysis using GC-MS, HPLC-EC and multiple reaction monitoring on small biomolecules such as 8-oxo-deoxyguanosine, homocysteine, and fluconazole. We also provide protein and peptide molecular weight analysis and sensitive protein identification service. We collaborate on proteomics projects to understand the mechanisms of carcinogenesis. Collaborative projects have successfully characterized protein modifications including chemical adducts, phosphorylation, acetylation, methylation and inhibitor binding sites. The core works to develop robust compound extraction and storage protocols for samples from tissues and biofluids. We test, implement and modify new services in response to researcher interests and innovations in mass spectrometry and sample preparation. The core serves as a resource for UT-Austin and CRED by providing high-end instrumentation and technical expertise as tools for advancing biological and biomedical research.

J Biomol Tech. 2009 February; 20(1): 83–84.

CF29-M The Vanderbilt Mass Spectrometry Shared Facilities

Abstract

The Vanderbilt Mass Spectrometry Research Center (MSRC) provides an integrated bioanalytical service facility to Vanderbilt researchers in addition to a comprehensive MS research component. The synergies achieved by merging research and service components provide investigators with state-of-the-art proteomics, tissue profiling/imaging and bioanalytical MS technologies that are more comprehensive than usually available in most academic laboratories. These cores are managed by a professional staff of six faculty members and nine research assistants, bioinformatics specialists and an instrument engineer. The Proteomics Laboratory supports multiple technology platforms, including HPLC peptide separations and 2D gel separations of intact proteins, followed by ESI-linear ion trap/orbitrap and MALDI-TOF/TOF MS and supporting bioinformatics for protein identification and targeted characterization. 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 expression studies on simple and complex proteomes. The tissue imaging core provides tissue sectioning, staining, and MS directly from tissue sections via either high resolution imaging across an entire tissue section, or higher-throughput histology-directed profiling using specific tissue areas, followed by biostatistical analysis of the MS data. 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 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.

J Biomol Tech. 2009 February; 20(1): 83–84.

CF30-M Automated Electron Transfer Dissociation of Large Peptides and Medium Size Proteins in a QTOF Instrument on an LC Timescale

Abstract

The combination of liquid chromatography (LC) and tandem mass spectrometric (MS) analysis of peptides produced by enzymatic digestion is an established technique for protein identification approaches. For the analysis of such biopolymers tandem mass spectrometers with electrospray ionization (ESI) are commonly used. Electron transfer dissociation (ETD) has become an important technique maintaining fragile modifications of biomolecules during the fragmentation process and is now well established on ion trap instruments. The high resolution and accuracy of orthogonal time-of-flight (oTOF) instruments will extend the possible applications to larger peptides and medium-size proteins. ETD in a hybrid quadrupole time-of-flight (QTOF) instrument has already been demonstrated. Major steps to enable LC applications of ETD in QTOF instruments will be presented: (i) the adaption of ETD reaction times to several kHz repetition rate of the TOF analyzer; (ii) automated precursor ion selection taking into account the charge state of the analyte ions, (iii) optimized ETD sequence timing. With this ETD QTOF prototype instrument the amino acid sequence of several peptides and small proteins were characterized.

J Biomol Tech. 2009 February; 20(1): 84.

CF32-M Fannie E. Rippel Biochemistry and Biotechnology Facility

Abstract

The Fannie E. Rippel Biochemistry and Biotechnology Facility (BBF) is a full-service, one-stop shopping, facility, from sample preparation to mass spectrometry, from biochemistry to data interpretation. A previous successful component, real-time PCR facility, has been moved to a genomics core. Technologies supported now include sample preparation, chromatography purification, metabolite and drug analytical chemistry, mass spectrometry, proteomics, and DNA synthesis.

J Biomol Tech. 2009 February; 20(1): 84.

CF33-M CBC/RRC Proteomics & Informatics Services Facility at UIC

Abstract

We will highlight services available at the CBC/RRC Proteomics and Informatics Services Facility (PISF), the core mass spectrometry facility for the Chicago area located at the University of Illinois at Chicago. Through funding from a grant from The Searle Funds at the Chicago Community Trust to the Chicago Biomedical Consortium we provide support to users from Northwestern University and the University of Chicago in addition to the University of Illinois at Chicago. Our instrumentation and services will be summarized with emphasis on new services and new equipment. We offer training to CBC users including a one-day overview and an annual four-day hands-on proteomics and informatics training course. We also host a CBC-sponsored monthly Proteomics Club for proteomics users in the Chicago area. Our facility offers self-serve instrument access for trained users in addition to routine samples and special project submission. OFFGel isoelectric focusing is provided as a service, along with MuDPIT, iTRAQ and other custom services. Please visit our website at http://proteomics.rrc.uic.edu/for additional details about our services, to submit samples, for recommended protocols for sample submission or for facility information formatted for grant submission. We’re available for consultation by phone, e-mail, Access Grid, or in person.

J Biomol Tech. 2009 February; 20(1): 84.

CF34-M iTraq Labeled Protein Quantification Using the TSQ Vantage LC-MS/MS

Abstract

Accurate quantification of proteins and peptides prior to analysis on sensitive mass spectrometers (MS) can be very useful in many proteomics applications—such as spectral counting and differential mass spectrometry (dMS). Accurate quantification of protein digests prior to MS analysis allows for proper loading of the sample and can prevent MS system damage or column overloading. Current methods of quantifying protein digests prior to MS analysis, such as near and far UV, fluorescence labeling, and dye assays, are very limited and are effective for proteins but not protein digests. Amino acid analysis (AAA) is a very common and reliable method of quantifying peptides/proteins. Traditional ion exchange amino acid analysis, however, requires a relatively large amount of sample (~2 nmol) and can take some time to yield results. More sensitive pre-column derivatization AAA can be performed but still offers only a small increase in sensitivity and is subject to matrix effects during derivatization. Because most proteomics experiments are sample limited, it is important to be able to quantify proteins and peptides using minute amounts of material. We will compare AAA sensitivity and accuracy using a triple quadrapole mass spectrometer (combined with Applied Biosystems iTraq reagents and Michrom Bioresources ADVANCE Plug-and-Play nano-spray source) to traditional post-column ninhydrin AAA. Our facility possesses Hitachi high-performance amino acid analyzers and over 20 years of AAA experience—allowing us to quickly and confidently verify the accuracy of the MS protein/peptide quantification.

J Biomol Tech. 2009 February; 20(1): 84–85.

CF35-M RI-INBRE Centralized Research Core Facility

Abstract

The RI-INBRE Centralized Research Core Facility inaugurated in July 2003, is being supported by the Rhode Island IDeA Network of Biomedical Research Excellence (RI-INBRE) grant and by the participating institutions that include: University of Rhode Island, Brown University, Rhode Island College, Providence College, Salve Regina University, and Roger Williams University. This facility is located in the College of Pharmacy at the University of Rhode Island’s Kingston campus. The Core Facility is providing access to research instrumentation and training support to RI-INBRE participants as well as other scientists affiliated with academic institutions and the private sector throughout the state of Rhode Island. Instrument reservation and scheduling are available, through the core facility’s website (wwww.uri.edu/inbre/corelab), on a first come, first served basis. All new users are supported with operator assisted access to the equipment. Independent access to the equipment is also available to all trained users. In addition, full service access via sample submission is provided particularly in ICP-MS, LC/MS/MS and N-terminal protein sequencing. After-hours access is granted through the University of Rhode Island I.D. card or by use of a key code password. This poster will present a detailed listing of Core Facility equipment and services. Supported by NIH-NCRR Grant # 1P20RR16457

J Biomol Tech. 2009 February; 20(1): 85.

CF36-M Instrumentation for Core Facilities: the NIH SIG and HEI Programs

Abstract

For many years, the NIH has provided expensive state of the art equipment to the biomedical community through the NCRR Shared Instrumentation Grant (SIG) and more recently, the High-End Instrumentation (HEI) programs. In many cases, these instruments are located in institutional core facilities which provide access and the high level technical expertise in cutting edge technologies and complex analytical procedures required for both basic and translational studies. Although the SIG and HEI programs fund instrumentation that span the technology spectrum, some instruments are placed in DNA sequencing, microarray and mass spectrometry cores managed by facility directors who are members of the ABRF. This talk will summarize the funding levels and trends in equipment for both the SIG and HEI programs.

J Biomol Tech. 2009 February; 20(1): 85.

CF37-M The Hartwell Center

Abstract

The Hartwell Center is a unique integration of high-throughput biotechnology resources, bioinformatics resources, and computing resources; designed to provide state-of-the-art tools for biomedical discovery. The Biotechnology laboratories provide cutting edge services in four areas: Macromolecular Synthesis, DNA Sequencing, Functional Genomics and Proteomics. They are supported by a staff of 30, including 9 Ph.D.-level scientists with expertise in all disciplines encompassed by the Center. The Macro-molecular Synthesis group provides a variety of research reagents via automated synthesis of oligonucleotides and peptides using standard and custom chemistries. The DNA sequencing laboratories provide investigators access to genome-wide sequence analysis using Roche FLX and Illumina technologies, as well as Sanger DNA sequence analysis, fragment size analysis, and SNP detection technologies. The Functional Genomics group provides microarray analysis services using commercial and custom-built arrays. The Proteomics group offers a range of protein chemistry capabilities including protein identification, intact mass measurement, peptide modification analysis, and molecular interaction analyses by surface and solution-based technologies. All of these resources are highly integrated and readily accessible using our Web-based on-line ordering, tracking, invoicing, and data retrieval system. Research Informatics within Information Sciences provides crucial bioinformatics and computing resources that support the high throughput data generated by the Hartwell Center and other technologies. This group includes 10 Ph.D.-level bioinformatics scientists and five staff members that maintain and operate a high-performance computing facility. Computational resources include an 810 CPU Linux cluster and more than 100 physical and virtual servers with 368 TB of disk storage. These computer systems provide access to a wide range of bioinformatics applications for genomic, proteomic and structural biology analyses. The wealth of resources offered by the Hartwell Center provide a broad range of chemistry, biomolecular, and bioinformatics services in support of research programs at St. Jude Children’s Research Hospital.

J Biomol Tech. 2009 February; 20(1): 85.

CF38-M The University of Oklahoma Health Sciences Center Laboratory for Genomics and Bioinformatics

Abstract

The University of Oklahoma Health Sciences Center’s Laboratory for Genomics and Bioinformatics (LGB) is a full service facility offering a wide range of services for DNA sequencing (custom and genome-scale projects), genotyping, fragment analysis, gene expression (real-time PCR and microarrays), protein analysis and bioinformatics. The LGB welcomes sample submission from any academic or industrial entity and we offer easy online submission and data retrieval via our facility webpage (http://microgen.ouhsc.edu). The laboratory is well equipped with the necessary robotics to serve customers requiring high-throughput services for PCR setup, plasmid preparation, and sample normalization in addition to the specialized services listed above. Available equipment includes two ABI 3730xl sequencers, an ABI SOLiD system, BiaCore T100, ABI 7500 Fast qPCR machine, Beckman NX liquid handler, an Agilent BioAnalyzer, and a Beckman PF2D Proteome lab and PA800 for protein analysis needs. In addition to the wet bench techniques we also offer bioinformatics support including database development and maintenance, custom script writing, data processing and analysis, and the development of Web based tools. To date, we have completed or are participating in the whole genome sequencing of 18 microorganisms (most are pathogens of human or agricultural importance), draft sequencing of 4 additional microbes, and EST sequencing. We also have provided microarray hybridization and data analyses for several gene expression studies in a variety of organisms, including human, mouse, and several microorganisms.

J Biomol Tech. 2009 February; 20(1): 86.

CF39-M Core Facility and Outreach Integration through the Vermont Genetics Network

Abstract

The Vermont Genetics Network (VGN) is funded by the National Center for Research Resources (NCRR), and is part of a National Institutes of Health (NIH) initiative called IDeA Networks of Biomedical Research Excellence (INBRE) to build biomedical research infrastructure. The VGN Program developed three core facilities at the University of Vermont (UVM); microarray, bioinformatics and proteomics. UVM acts as the lead institute in a partnership with five baccalaureate colleges throughout Vermont. VGN outreach develops educational modules based on the technologies available in the VGN sponsored cores. We use our outreach program to inspire Vermont undergraduates within and outside our partner colleges 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. This program has lead 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 the state. We are seeing this goal being achieved with subsequent deliveries at several colleges. The bioinformatics outreach tutorial was developed in 2006 in association with Dr. William Barnes on sabbatical from Clarion University of Pennsylvania. The module has been delivered by the outreach program since the Fall of 2007. This module is also being quickly integrated into the baccalaureate colleges’ curriculum. The proteomics module is schedules for beta testing in the spring of 2009 at UVM 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 helps to bring about sustainable changes in how we, in Vermont, carry out research and educate our next generation of scientists.

J Biomol Tech. 2009 February; 20(1): 86.

CF40-M Proteomics and Glycomics at the Australian Proteome Analysis Facility (APAF)

Abstract

Whilst a PhD student at APAF in 1995, Marc Wilkins first coined the fusion word proteomics. Since then APAF has been a leading institution in developing technology and providing proteomics services to the local Australian and international community. Traditionally strong in 2D gel electrophoresis and classical protein chemistry (such as Edman sequencing and amino acid analysis) APAF has evolved with the growth of proteomics and now provides a comprehensive range of proteomic MS-based techniques with particular focus on biomarker discovery through quantitative proteomics, MRMs and more recently glycomics services.

APAF has considerable experience in quantifying differences in protein expression by iTRAQ labelling and differential fluorescence gel electrophoresis (DIGE). For quantitation of targeted proteins in complex mixtures multiple reaction monitoring (MRM) analysis has been developed. Expertise is also available in label-free quantitation and peptide IPG-IEF “shotgun” methodologies. As well, we offer routine MS analyses to identify and sequence proteins from any biological source. Antibody-based removal of abundant proteins from samples, such as plasma, is available as a sample preparation technology. The importance of post-translational modifications (PTMs) is well recognised in enabling proteins to achieve full biological function. The most common, and largely ignored, PTM is glycosylation. Due to a recent investment in glycomics APAF now offers N- and O-linked glycosylation analysis by MS. This is proving particularly useful for characterising recombinant proteins and antibodies and supporting the regulatory approval process for biotech products. APAF has state-of-the-art, user-friendly facilities in mass spectrometry, gel electrophoresis, chromatography and bioinformatics. These provide competitive, high quality services to the scientific community. We have broad experience in a wide range of samples including the more difficult to analyse biofluids, such as plasma and urine, and plants proteomes like the wheat glutenins. The facility is available to researchers under access arrangements. Further details at www.proteome.org.au.

J Biomol Tech. 2009 February; 20(1): 86.

CF41-M Core Synthesis Facility at NDSU: Research and Capabilities

Abstract

The Core Synthesis Facility (CSF) was established as a part of the Center for Protease Research at NDSU in April 2008 with funding from an NCRR-NIH-COBRE grant. The primary objective of CSF is to act as a strong in-house support system to facilitate biomedical researchers in accomplishing their research goals. The CSF is equipped with modern synthetic technologies and state of the art instrumentation. The research in the CSF is focused on organic synthesis of small molecules for biomedical applications and analytical characterization of substrates of interest. The current research projects involve synthesis of enzyme inhibitors, fluorescent labeling of peptides and amino acids, development of a cytotoxic drug delivery model and qualitative analysis of natural products by LC-MS/HRMS. The CSF also offers scientific consultation and provides student training. On a whole, the research in the CSF is dedicated towards the treatment of a variety of diseases and our future goal is to establish a nationwide clientele.

J Biomol Tech. 2009 February; 20(1): 87.

CF43-M Microscopy and Imaging Facility

Abstract

The Microscopy and Imaging Facility of the Cornell University Life Sciences Core Laboratories Center (CLC) provides an array of shared research resources and services relating to optical microscopy, including confocal microscopy, fluorescence imaging, spectrofluorimetry, ultrasound, image processing and analysis. The mission of the facility is to provide cutting edge technologies and high quality services that will significantly contribute to life sciences research, training and education programs.

J Biomol Tech. 2009 February; 20(1): 87.

CF44-M Laser Capture Microdissection

Abstract

The state-of-the-art technology laser capture microdissection (LCM) provides the researcher with a means to isolate a pure population of cells from heterogeneous tissue specimens. The purified DNA, RNA or protein from captured cells can be used for a wide range of downstream applications such as gene expression analysis, loss of heterozygosity studies, or proteomic assays, etc. Many different types of molecular analyses have been successfully performed on cells procured by LCM and a number of new LCM technologies have been developed in our LCM facility, which will contribute to meeting the growing interdisciplinary research program at Fox Chase Cancer Center and potentially other ABRF members.

J Biomol Tech. 2009 February; 20(1): 87.

CF45-M The UC Davis Genome Center Proteomics Facility

Abstract

The UC Davis Genome Center Proteomics Facility offers protein analysis via amino acid analysis, Edman sequencing and proteomic services including protein identification, post-translational modification discovery, label free quantitation and targeted protein analysis. The facility utilizes state of the art instrumentation, including a Thermo LTQ-FT Ultra, and TSQ Vantage mass spectrometers. Our group utilizes open source software and uses techniques and protocols that are made publicly available. Using open source software allows users to analyze their own data without 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

J Biomol Tech. 2009 February; 20(1): 87.

CF46-M Computational Biology Service Unit: Microsoft High Performance Computing Institute

Abstract

The Computational Biology Service Unit (CBSU) of the Cornell University Life Sciences Core Laboratories Center (CLC) provides bioinformatics and computational biology resources and services to the university community and to outside investigators. The CBSU is a Microsoft designated High Performance Computing Institute. Facility resources include a set of servers and compute clusters with over 1200 cores. The goal of the facility is to meet the increasing need of Cornell investigators for broad support in computational biology and bioinformatics.

J Biomol Tech. 2009 February; 20(1): 87.

CF47-M IDPicker 2.0: Improved Protein Assembly with High Discrimination Peptide Identification Filtering

Abstract

Tandem mass spectrometry-based shotgun proteomics has become a widespread technology for analyzing complex protein mixtures. A number of database searching algorithms have been developed to assign peptide sequences to tandem mass spectra. Assembling the peptide identifications to proteins, however, is a challenging issue because many peptides are shared by multiple proteins. IDPicker is an open-source protein assembly tool that derives a minimum protein list from peptide identifications filtered to a specified False Discovery Rate. Here we update IDPicker to increase confident peptide identifications by combining multiple scores produced by database search tools. By segregating peptide identifications on both charge state and the number of tryptic termini in validation, IDPicker yields more confident identifications for protein assembly. The new version achieves higher accuracy by requiring additional novel peptides in the parsimony process, especially when searching against multiple-species databases. IDPicker has been tuned for incorporation in many identification workflows by the addition of a graphical user interface and the ability to read identifications from the pepXML format. These advances position IDPicker for high peptide discrimination, reliable protein assembly, and highly-scalable proteomics studies.

J Biomol Tech. 2009 February; 20(1): 88.

CF48-M Extending MALDI-QqQ-MS Enzyme Screening Assays to Targets with Small Molecule Substrates

Abstract

Mass spectrometry-based high throughput screening has tremendous future potential as an alternative to current screening methods due to its speed, sensitivity, reproducibility and label-free readout. In addition, this method offers a direct readout of the substrate and product, thus minimizing the potential for false positive or false negative hits. We recently reported that a new generation matrixassisted laser desorption ionization-triple quadrupole mass spectrometer (MALDI-QqQ-MS) is ideally suited for a variety of enzyme assays and screening protocols. This instrument provides comparable speeds (at greater than a sample per second) with superior signal-to-background, better reproducibility and a reagent cost savings of greater than 90% as compared to typical fluorescence-coupled assays. Thus far the MALDI-based readout has been validated for a variety of enzyme classes (kinases, phosphatases, proteases, hydroxylases), however all these targets have peptide substrates that are readily monitored without interference from the MALDI matrix. To further extend the application of the MALDI-QqQ readout to enzymes with small molecule, non-peptide substrates, we evaluated this method for measuring enzyme activity and inhibition of acetylcholinesterase. Due to matrix interference in measuring these small molecules during the MALDI process, multiple reaction monitoring (MRM), available on the QqQ instruments, was used to generate a selective MS/MS transition and accurately measure both the substrate (acetylcholine) and the product (choline) of acetylcholinesterase. Importantly, accurate dose-dependant inhibition measurements were also demonstrated thus validating the MRM readout for enzymes with small molecule substrates and products. Collectively, these data demonstrate that a MALDI-QqQ-MS based readout platform is amenable for small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs.

J Biomol Tech. 2009 February; 20(1): 88.

CF49-M High Mobility Group Box 1 (HMGB1) In Eosinophil Activation

Abstract

Eosinophils have been implicated in allergic inflammation and certain parasitic and viral infections. Our proteomic studies of non-activated and activated eosinophils identified the expression and release of HMGB1. HMGB1 plays a prominent role in immunoregulatory cell activation including granulocyte activation. Our experiments were designed to determine whether eosinophil-derived HMGB1 contributes to the autocrine activation of human peripheral blood eosinophils. HMGB1-presence in eosinophils was determined using western blotting and two-dimensional gel electrophoresis. Pro-Q Diamond phosphoprotein stain and Sypro Ruby protein stain were used for detection of phosphoproteins or proteins, respectively. Cell survival rates and expression of CD69 were determined after stimulation of eosinophils with GM-CSF, rHMGB1, and various HMGB1 inhibitors. Eosinophils stimulated with either GM-CSF, rHMGB1, and eosinophil-derived HMGB1 showed significantly higher viability and expression of CD69. Pre-treating eosinophils with glycyrrhizin, a specific inhibitor of HMGB1 activity, partially inhibited prolongation of survival as well as upregulation of CD69. This upregulation was significantly inhibited by treatment with glycyrrhizin or anti-RAGE2 antibody. The phosphoproteomic profile of HMGB1 stimulated eosinophils also changed. The phosphoproteomic pattern and the proteomic pattern of eosinophils treated with HMGB1 and GM-CSF vary as compared to control cells and compared to each other. Our studies demonstrate the autocrine activation of eosinophils through HMGB1. These findings indicate a significant immunoregulatory role for eosinophils and provide a novel mechanism for the characterization of eosinophil-associated pathologies. This study was supported by the NIH/NHLBI Proteomics Initiative NO1-HV-28184 (AK).

J Biomol Tech. 2009 February; 20(1): 88–89.

CF50-M Using Targeted Proteomics to Assess the Impact of Sirtuins on Liver Metabolism in Obesity

Abstract

The NAD-dependent deacetylase SirT1 regulates lipid and glucose metabolism in liver and increased SirT1 activity in caloric-restricted models has been linked to extended life span in several species. Deacetylation of transcription factors and co-modulators, such as p53, NF-κB, C/EPBβ and PGC-1, allows SirT1 to sense and regulate energy levels. The SirT family can also deacetylate mitochondrial proteins, suggesting that posttranslational modification by sirtuins may have global effects on energy metabolism especially gluconeogenesis and lipogenesis. Livers from mice fed either control or a high fat (HF) diet (45 kcal % as fat, 12 wks) were harvested and homogenized. SirT1 levels were measured in whole cell lysates by Western blot. Proteins modified by lysine acetylation were immunoprecipitated with anti-acetyllysine antibody and subsequently separated by one dimensional gel electrophoresis. Bands showing differential staining between the control and HF fed mice were excised and proteins were digested with trypsin. Tandem mass spectrometry using an Agilent Ultra quadrupole ion trap generated product ion spectra that were searched with SpectrumMill against the SwissProt database. The levels and acetylation of identified proteins were validated by immunoprecipitation and Western blotting. Mice on the HF diet were obese, with fatty livers and reduced SirT1 activity as assessed by the NAD+/NADH ratio. SirT1 protein expression levels did not significantly change, however. Acetylation of a subset of the proteins identified, such as carbamoyl-phosphate synthase, uricase, pyruvate carboxylase and ATP synthase, has been previously reported. Interestingly, peroxiredoxin, catalase, and Hsp70, proteins involved in redox and the stress response, were hyperacetylated in the livers of obese mice. We postulate that modification of those proteins could influence the ability of obese mice to modulate oxidative stress, gluconeogenesis, and lipogenesis. Globally surveying lysine acetylated proteins using immunoprecipitation and gel electrophoresis/tandem mass spectrometry provides insights into how obesity impacts liver metabolism.


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