Multiple Reaction Monitoring-Based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma

University of Victoria-Genome British Columbia Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada.
Molecular & Cellular Proteomics (Impact Factor: 6.56). 06/2009; 8(8):1860-77. DOI: 10.1074/mcp.M800540-MCP200
Source: PubMed


Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([(13)C(6)]Arg or [(13)C(6)]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.

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    • "Forty-three proteotypic tryptic peptides (corresponding to 43 plasma proteins) were originally selected using bioinformatics. Synthesis of C-terminal [ 13 C] and/or [ 15 N]-labeled analogues of these proteotypic peptides was performed at the University of Victoria (UVic)-Genome British Columbia Proteomics Centre using Fmoc protection chemistry [17] on a Prelude or an Overture Robotic Peptide Synthesizer (Peptide Technologies; Seattle, WA, USA). After synthesis, the SIS peptides were purified by reversedphase HPLC using an Agilent 1260 Infinity LC, with the peptides' identities being subsequently verified by MALDI-TOF-MS on an Ultraflex III mass spectrometer (Bruker Daltonics; Bremen, Germany). "
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    ABSTRACT: The reproducibility of plasma protein quantitation between laboratories and between instrument types was examined in a large-scale international study involving 16 laboratories and 19 LC–MS/MS platforms, using two kits designed to evaluate instrument performance and one kit designed to evaluate the entire bottom-up workflow. There was little effect of instrument type on the quality of the results, demonstrating the robustness of LC/MRM-MS with isotopically labeled standards. Technician skill was a factor, as errors in sample preparation and sub-optimal LC–MS performance were evident. This highlights Please cite this article in press as: A.J. Percy, et al., Inter-laboratory evaluation of instrument platforms and experimental workflows for quantitative accuracy and reproducibility assessment, EuPA Open Proteomics (2015), http://dx.
    Full-text · Article · Jun 2015 · EuPA Open Proteomics
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    • "Hence, there is a need to develop high-throughput assays with simple sample preparation and reduced MS analysis time. Multiple reaction monitoring (MRM) is a tandem MS (MS/MS) scan mode unique to triple quadrupole MS instrumentation that is capable of rapid, sensitive, and specific quantitation of peptides in highly complex sample matrices, such as plasma [9] [10]. MRM is a targeted approach that requires knowledge of the molecular weight the peptide of interest and its fragmentation pattern, leading to the generation of target " transitions " for monitoring protein levels. "
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    • "using solid phase peptide synthesis (SPPS), as previously described [35]. "
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