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 ( MS is capable of sensitive and accurate protein quantitation based on the quantitation of proteolytic peptides as surrogates for the corresponding intact proteins. Over the past 10 years, MS-based protein quantitation based on the analysis of peptides (in other words, based on "bottom-up" proteomics) has had a profound impact on how biological problems can be addressed (1, 2). Although advances in MS instrumentation have contributed to the improvement of MS-based protein quantitation, the use of stable isotopes in quantitative work flows has arguably had the greatest impact in improving the quality and reproducibility of MS-based protein quantitation (3-5).The ongoing development of untargeted MS-based quantitation work flows has focused on increasingly exhaustive sample prefractionation methods, at both the protein and peptide levels, with the goal of detecting and quantifying entire proteomes (6). Although untargeted MS-based quantitation work flows have their utility, they are costly in terms of lengthy MS data acquisition and analysis times, and as a result, they are often limited to quantifying differences between small sample sets (n Ͻ 10). To facilitate rapid quantitation of larger, clinically relevant sample sets (n Ͼ 100) there is a need to both simplify sample preparation and reduce MS analysis time.Multiple reaction monitoring (MRM) 1 is a tandem MS (MS/MS) scan mode unique to triple quadrupole MS instrumentation that is capable of rapid, sensitive, and specific quantitation of analytes in highly complex sample matrices (7). MRM is a targeted approach that requires knowledge of the molecular weight of an 1 The abbreviations used are: MRM, multiple reaction monitoring; CE, collision energy; CV, coefficient of variation; CZE, capillary zone electrophoresis; DP, declustering potential; LOQ, limit of quantitation; Q1, quadrupole 1; Q3, quadrupole 3; SIS, stable isotope-labeled standard; XIC, extracted ion chromatogram; iTRAQ, isobaric tags for relative and absolute quantitation. Research

Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides from 45 moderate- to high-abundance plasma proteins, using tandem mass spectrometry in multiple reaction monitoring mode, with a mixture of stable-isotope labeled analogues of these proteotypic peptides as internal standards. When the digestion efficiencies of these 14 methods were compared, we found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol. SDS, however, can be a serious interference for subsequent mass spectrometry. DOC, on the other hand, can be easily removed from the samples by acid precipitation. Examining the results of a reproducibility study, done with 5 replicate digestions, DOC and SDS with a 9 h digestion time produced the highest average digestion efficiencies (~80%), with the highest average reproducibility (<5% error, defined as the relative deviation from the mean value). However, because of potential interferences resulting from the use of SDS, we recommend DOC with a 9 h digestion procedure as the optimum protocol.

A highly-multiplexed MRM-based assay for determination of cardiovascular disease (CVD) status and disease classification has been developed for clinical research. A high-flow system using ultra-high performance LC and an Agilent 6490 triple quadrupole mass spectrometer, equipped with an ion funnel, provided ease of use and increased the robustness of the assay. The assay uses 135 stable isotope-labeled peptide standards for the quantitation of 67 putative biomarkers of CVD in tryptic digests of whole plasma in a 30-min assay. Eighty-five analyses of the same sample showed no loss of sensitivity (<20% CV for 134/135 peptides) and no loss of retention time accuracy (<0.5% CV for all peptides). The maximum linear dynamic range of the MRM assays ranged from 10(3) -10(5) for 106 of the assays. Excellent linear responses (r >0.98) were obtained for 117 of the 135 peptide targets with attomole level limits of quantitation (<20% CV and accuracy 80-120%) for 81 of the 135 peptides. The assay presented in this study is easy to use, robust, sensitive, and has high-throughput capabilities through short analysis time and complete automated sample preparation. It is therefore well suited for CVD biomarker validation and discovery in plasma.