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1.  Multiplexed quantification of estrogen receptor and HER2/Neu in tissue and cell lysates by peptide immunoaffinity enrichment mass spectrometry 
Proteomics  2012;12(8):1253-1260.
Access to a wider range of quantitative protein assays would significantly impact the number and use of tissue markers in guiding disease treatment. Quantitative mass spectrometry-based peptide and protein assays, such as immuno-SRM assays, have seen tremendous growth in recent years in application to protein quantification in biological fluids such as plasma or urine. Here, we extend the capability of the technique by demonstrating the application of a multiplexed immuno-SRM assay for quantification of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) levels in cell line lysates and human surgical specimens. The performance of the assay was characterized using peptide response curves, with linear ranges covering approximately 4 orders of magnitude and limits of detection in the low fmol/mg lysate range. Reproducibility was acceptable with median coefficients of variation of approximately 10%. We applied the assay to measurements of ER and HER2 in well-characterized cell line lysates with good discernment based on ER/HER2 status. Finally, the proteins were measured in surgically resected breast cancers, and the results showed good correlation with ER/HER2 status determined by clinical assays. This is the first implementation of the peptide-based immuno-SRM assay technology in cell lysates and human surgical specimens.
doi:10.1002/pmic.201100587
PMCID: PMC3418804  PMID: 22577026
Estrogen receptor; HER2/Neu; immunoaffinity; peptides; tissue
2.  Peptide immunoaffinity enrichment coupled with mass spectrometry for peptide and protein quantification 
Clinics in laboratory medicine  2011;31(3):385-396.
Synopsis
Quantitative protein assays are needed in a wide range of biological studies. Traditional immunoassays are not available for a large number of proteins and development of new immunoassays requires a significant investment in time and money. The development of assays using peptide immunoaffinity enrichment coupled with targeted mass spectrometry has many advantages including versatility in design, ease of use, enhanced specificity, and good performance characteristics. This review presents recent developments in the characterization and implementation of immuno-SRM assays.
doi:10.1016/j.cll.2011.07.004
PMCID: PMC3186071  PMID: 21907104
quantitative proteomics; selected reaction monitoring; stable isotope dilution; SISCAPA; immuno-SRM; immuno-mass spectrometry
3.  Proteome and Transcriptome Profiles of a Her2/Neu-driven Mouse Model of Breast Cancer 
Proteomics. Clinical applications  2011;5(3-4):179-188.
Purpose
We generated extensive transcriptional and proteomic profiles from a Her2-driven mouse model of breast cancer that closely recapitulates human breast cancer. This report makes these data publicly available in raw and processed forms, as a resource to the community. Importantly, we previously made biospecimens from this same mouse model freely available through a sample repository, so researchers can obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens.
Experimental design
Twelve datasets are available, encompassing 841 LC-MS/MS experiments (plasma and tissues) and 255 microarray analyses of multiple tissues (thymus, spleen, liver, blood cells, and breast). Cases and controls were rigorously paired to avoid bias.
Results
In total, 18,880 unique peptides were identified (PeptideProphet peptide error rate ≤1%), with 3884 and 1659 non-redundant protein groups identified in plasma and tissue datasets, respectively. Sixty-one of these protein groups overlapped between cancer plasma and cancer tissue.
Conclusions and clinical relevance
These data are of use for advancing our understanding of cancer biology, for software and quality control tool development, investigations of analytical variation in MS/MS data, and selection of proteotypic peptides for MRM-MS. The availability of these datasets will contribute positively to clinical proteomics.
doi:10.1002/prca.201000037
PMCID: PMC3069718  PMID: 21448875
Breast cancer; Her2; mouse; proteome; transcriptome
4.  A targeted proteomics–based pipeline for verification of biomarkers in plasma 
Nature biotechnology  2011;29(7):625-634.
High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.
doi:10.1038/nbt.1900
PMCID: PMC3232032  PMID: 21685906
5.  Effect of Collision Energy Optimization on the Measurement of Peptides by Selected Reaction Monitoring (SRM) Mass Spectrometry 
Analytical chemistry  2010;82(24):10116-10124.
Proteomics experiments based on Selected Reaction Monitoring (SRM, also referred to as Multiple Reaction Monitoring or MRM) are being used to target large numbers of protein candidates in complex mixtures. At present, instrument parameters are often optimized for each peptide, a time and resource intensive process. Large SRM experiments are greatly facilitated by having the ability to predict MS instrument parameters that work well with the broad diversity of peptides they target. For this reason, we investigated the impact of using simple linear equations to predict the collision energy (CE) on peptide signal intensity and compared it with the empirical optimization of the CE for each peptide and transition individually. Using optimized linear equations, the difference between predicted and empirically derived CE values was found to be an average gain of only 7.8% of total peak area. We also found that existing commonly used linear equations fall short of their potential, and should be recalculated for each charge state and when introducing new instrument platforms. We provide a fully automated pipeline for calculating these equations and individually optimizing CE of each transition on SRM instruments from Agilent, Applied Biosystems, Thermo-Scientific and Waters in the open source Skyline software tool (http://proteome.gs.washington.edu/software/skyline).
doi:10.1021/ac102179j
PMCID: PMC3005404  PMID: 21090646
6.  Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry 
There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.
doi:10.3791/2812
PMCID: PMC3197439  PMID: 21841765
7.  Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring-mass spectrometry 
Journal of immunological methods  2009;353(1-2):49-61.
Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) utilizes antibodies to enrich peptides from complex matrices for quantitation by stable isotope dilution mass spectrometry. SISCAPA improves sensitivity and limits the sample handling required for plasma-based analysis. Thus far, SISCAPA assays have been performed using polyclonal antibodies, yet monoclonal antibodies are an attractive alternative since they provide exquisite specificity, a renewable resource, and the potential for isolation of clones with very high affinities (10-9 M or better). The selection of a good monoclonal antibody out of hundreds-to-thousands of clones presents a challenge, since the screening assay should ideally be in the format of the final SISCAPA assay, but performing the assays manually is labor- and time-intensive. In this manuscript, we demonstrate that monoclonal antibodies can be used in SISCAPA assays, and we describe an automated high-throughput SISCAPA method that makes screening of large numbers of hybridomas feasible while conserving time and resources.
doi:10.1016/j.jim.2009.11.017
PMCID: PMC2839902  PMID: 19961853
SISCAPA; anti-peptide antibody; monoclonal antibody screening; selected reaction monitoring-mass spectrometry; automation; ELISA
8.  The interface between biomarker discovery and clinical validation: The tar pit of the protein biomarker pipeline 
Proteomics. Clinical applications  2008;2(10-11):1386-1402.
The application of “omics” technologies to biological samples generates hundreds to thousands of biomarker candidates; however, a discouragingly small number make it through the pipeline to clinical use. This is in large part due to the incredible mismatch between the large numbers of biomarker candidates and the paucity of reliable assays and methods for validation studies. We desperately need a pipeline that relieves this bottleneck between biomarker discovery and validation. This paper reviews the requirements for technologies to adequately credential biomarker candidates for costly clinical validation and proposes methods and systems to verify biomarker candidates. Models involving pooling of clinical samples, where appropriate, are discussed. We conclude that current proteomic technologies are on the cusp of significantly affecting translation of molecular diagnostics into the clinic.
doi:10.1002/prca.200780174
PMCID: PMC2957839  PMID: 20976028
Biomarker verification; Multiple reaction monitoring; Targeted proteomics
9.  The evolving role of mass spectrometry in cancer biomarker discovery 
Cancer biology & therapy  2009;8(12):1083-1094.
Although the field of mass spectrometry-based proteomics is still in its infancy, recent developments in targeted proteomic techniques have left the field poised to impact the clinical protein biomarker pipeline now more than at any other time in history. For proteomics to meet its potential for finding biomarkers, clinicians, statisticians, epidemiologists and chemists must work together in an interdisciplinary approach. These interdisciplinary efforts will have the greatest chance for success if participants from each discipline have a basic working knowledge of the other disciplines. To that end, the purpose of this review is to provide a nontechnical overview of the emerging/evolving roles that mass spectrometry (especially targeted modes of mass spectrometry) can play in the biomarker pipeline, in hope of making the technology more accessible to the broader community for biomarker discovery efforts. Additionally, the technologies discussed are broadly applicable to proteomic studies, and are not restricted to biomarker discovery.
PMCID: PMC2957893  PMID: 19502776
targeted proteomics; multiple reaction monitoring; selected reaction monitoring; biomarker; mass spectrometry
10.  Antibody-based enrichment of peptides on magnetic beads for mass spectrometry-based quantification of serum biomarkers 
Analytical biochemistry  2006;362(1):44-54.
A major bottleneck for validation of new clinical diagnostics is the development of highly sensitive and specific assays for quantifying proteins. We previously described a method, Stable Isotope Standards with Capture by Anti-Peptide Antibodies, wherein a specific tryptic peptide is selected as a stoichiometric representative of the protein from which it is cleaved, is enriched from biological samples using immobilized antibodies, and is quantitated using mass spectrometry against a spiked internal standard to yield a measure of protein concentration. In this report, we optimize a magnetic bead-based platform amenable to high throughput peptide capture and demonstrate that antibody capture followed by mass spectrometry can achieve ion signal enhancements on the order of 103, with precision (CVs <10%) and accuracy (relative error ~20%) sufficient for quantifying biomarkers in the physiologically relevant ng/mL range. These methods are generally applicable to any protein or biological fluid of interest and hold great potential for providing a desperately needed bridging technology between biomarker discovery and clinical application.
doi:10.1016/j.ab.2006.12.023
PMCID: PMC1852426  PMID: 17241609
Biomarkers; Anti-peptide antibody; LC/MS/MS; Targeted proteomics; Stable isotope dilution; Peptide quantitation
11.  Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography—Tandem Mass Spectrometry 
The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35–60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.
doi:10.1021/pr9006365
PMCID: PMC2818771  PMID: 19921851
12.  Performance Metrics for Liquid Chromatography-Tandem Mass Spectrometry Systems in Proteomics Analyses* 
A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.
doi:10.1074/mcp.M900223-MCP200
PMCID: PMC2830836  PMID: 19837981

Results 1-12 (12)