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MRM ‐based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma
Author(s) -
Domanski Dominik,
Percy Andrew J.,
Yang Juncong,
Chambers Andrew G.,
Hill John S.,
Freue Gabriela V. Cohen,
Borchers Christoph H.
Publication year - 2012
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100568
Subject(s) - multiplex , chromatography , triple quadrupole mass spectrometer , chemistry , selected reaction monitoring , mass spectrometry , peptide , biomarker , biomarker discovery , linear range , detection limit , tandem mass spectrometry , biology , proteomics , bioinformatics , biochemistry , gene
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 A gilent 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.