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

• DOI: 10.1074/mcp.m800540-mcp200
• Publication Date: 2009-05-03T00:12:54Z
• Authors: Michael A. Kuzyk; Derek Smith; Juncong Yang; Tyra J. Cross; Angela M. Jackson; Darryl B. Hardie; N. Leigh Anderson; Christoph H. Borc...
• Keywords: Bottom-up proteomics; Peptide mass fingerprinting; Coefficient of variation; Standard curve; Quantitative Analysis

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