Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma
ABSTRACT 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  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). "
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.
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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  . 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. "
ABSTRACT: The oxidative modification of apolipoprotein A-I’s methionine148 (M148) is associated with defective HDL function in vitro. Multiple reaction monitoring (MRM) is a mass spectrometric technique that can be used to quantitate post-translational modifications. In this study, we developed an MRM assay to monitor the abundance ratio of the peptide containing oxidized M148 to the native peptide in ApoA-I. Measurement of the oxidized-to-unoxidized-M148 ratio was reproducible (CV < 5%). The extent of methionine M148 oxidation in the HDL of healthy controls, and type 2 diabetic participants with and without prior cardiovascular events (CVD) were then examined. The results suggest a significant increase in the relative ratio of the peptide containing oxidized M148 to the unmodified peptide in the HDL of participants with diabetes and CVD (p < 0.001), compared to participants without CVD. Monitoring the abundance ratio of the peptides containing oxidized and unoxidized M148 by MRM provides a means of examining the relationship between M148 oxidation and vascular complications in CVD.Translational Proteomics 12/2014; 4-5. DOI:10.1016/j.trprot.2014.10.001
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- "using solid phase peptide synthesis (SPPS), as previously described . "
ABSTRACT: Plasma renin activity (PRA) is essential for the screening and diagnosis of primary aldosteronism (PA), a form of secondary hypertension, which affects approximately 100 million people worldwide. It is commonly determined by radioimmunoassay (RIA) and, more recently, by relatively low-throughput LC-MS/MS methods. In order to circumvent the negative aspects of RIAs (radioisotopes, cross-reactivity) and the low throughput of LC-MS based methods, we have developed a high-throughput immuno-MALDI (iMALDI)-based assay for PRA determination using an Agilent Bravo for automated liquid handling and a Bruker Microflex LRF instrument for MALDI analysis, with the goal of implementing the assay in clinical laboratories. The current assay allows PRA determination of 29 patient samples (192 immuno-captures), within ~6-7 hours, using a 3-hour Ang I generation period, at a 7.5-fold faster analysis time than LC-MS/MS. The assay is performed on 350μL of plasma, and has a linear range from 0.08 - 5.3ng/L/s in the reflector mode, and 0.04 - 5.3ng/L/s in the linear mode. The analytical precision is 2.0 - 9.7% CV in the reflector mode, and 1.5 - 14.3% CV in the linear mode. A method comparison to a clinically employed LC-MS/MS assay for PRA determination showed excellent correlation within the linear range, with an R(2) value of ≥0.98. This automated high throughput iMALDI platform has clinically suitable sensitivity, precision, linear range, and correlation with the standard method for PRA determination. Furthermore, the developed workflow based on the iMALDI technology can be used for the determination of other proteomic biomarkers. This article is part of a Special Issue entitled: Medical Proteomics. Guest Editors: Tadashi Kondo, Helmut Meyer, and Barbara Sitek. This article is part of a Special Issue entitled: Medical Proteomics. Copyright © 2014. Published by Elsevier B.V.Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 10/2014; 1854(6). DOI:10.1016/j.bbapap.2014.10.008 · 2.75 Impact Factor