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

Kuzyk MA, Smith D, Yang J, Cross TJ, Jackson AM, Hardie DB, Anderson NL and Borchers CH

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 cocktail of 45 peptide standards, easily adaptable to common plasma proteomics workflows, has been 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. SIS peptides were added immediately following tryptic digestion since addition of SIS 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 in order to generate the most abundant precursor ions and y-ion fragments. Concentrations of individual peptide standards in the cocktail 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% CV) 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 CV's 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 cocktail of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling since 31 of the 45 proteins are putative biomarkers of cardiovascular disease.