Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma
Open accessAbstract
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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Various LC-MS/MS assays for multiplexed quantification of apolipoproteins or phenotyping of apoE have been reported.
“Various LC-MS/MS assays for multiplexed quantification of apolipoproteins (14-23) or phenotyping of apoE (24-27) have been reported.”
From Automated multiplex LC-MS/MS assay for quantifying serum apolipoproteins A-I, B, C-I, C-II, C-III, and E with qualitative apolipoprotein E phenotyping (2016) · cites this paper as [22]
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- Authors
- Kuzyk, M. A., Smith, D., Yang, J., Cross, T. J., Jackson, A. M., Hardie, D. B., Anderson, N. L., & Borchers, C. H.
- Affiliations
- University of Victoria (CA)
- Corresponding author
- Borchers, Christoph H.
- Venue
- Molecular & Cellular Proteomics
- Publisher
- Elsevier BV
- Published
- 2009-08
- Volume
- 8
- Issue
- 8
- Pages
- 1860-1877
- ISSN
- 1535-9476 (ISSN-L)
- PMC
- PMC2722777
- OpenAlex
- W1974500649
- Cited by
- 515
- References
- 1
- Citations by year
- 2026: 8 · 2025: 7 · 2024: 7 · 2023: 13 · 2022: 10 · 2021: 11 · 2020: 21 · 2019: 19 · 2018: 20 · 2017: 30 · 2016: 23 · 2015: 46 · 2014: 51 · 2013: 62 · 2012: 85
- FWCI
- 25.20
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular & Cellular Proteomics (publishedVersion, cc-by)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Genome Canada
- Genome British Columbia
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- multiple reaction monitoring, human plasma, stable isotope-labeled peptides, trypsin digestion, cardiovascular biomarkers, biomarker discovery
- MeSH terms
- Blood Proteins, Chromatography, High Pressure Liquid, Humans, Male, Reference Standards, Mass Spectrometry, Trypsin, Reproducibility of Results, Electrophoresis, Capillary, Proteomics
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