1222 Proteomics 2012, 12, 1222–1243 DOI 10.1002/pmic.201100568 RESEARCH ARTICLE MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma Dominik Domanski 1 , Andrew J. Percy 1 , Juncong Yang 1 , Andrew G. Chambers 1 , John S. Hill 2,3 , Gabriela V. Cohen Freue 2 and Christoph H. Borchers 1,4 1 University of Victoria – Genome British Columbia Proteomics Centre, Victoria, BC, Canada 2 PROOF Centre of Excellence, Vancouver, BC, Canada 3 The James Hogg Research Centre St. Paul’s Hospital University of British Columbia & Institute for Heart + Lung Health, Vancouver, BC, Canada 4 Department of Biochemistry & Microbiology and University of Victoria – Genome BC Proteomics Centre University of Victoria, Victoria, BC, Canada 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. Keywords: Biomarker / Biomedicine / Cardiovascular disease / MRM / Plasma / Quantitation Received: October 31, 2011 Revised: December 20, 2011 Accepted: January 10, 2012 1 Introduction Cardiovascular disease (CVD) is the leading cause of morbid- ity and mortality in adults worldwide [1]. This chronic disease Correspondence: Dr. Christoph H. Borchers, Department of Bio- chemistry & Microbiology, University of Victoria–Genome BC Pro- teomics Centre, Victoria, BC V8Z 7X8, Canada E-mail: christoph@proteincentre.com Fax: +1-250-483-3238 Abbreviations: CE, collision energy; CVD, cardiovascular dis- ease; Fmoc, N-(9-fluorenyl)methoxycarbonyl; FWHM, full width half maximum; MRM, multiple reaction monitoring; SHBG, sex hormone-binding globulin; SIS, stable isotope-labeled internal standard; TCEP, tris(2-carboxyethyl)phosphine; UHPLC, ultra- high performance liquid chromatography; LCULQ, upper limit of quantitation; Q1, quadrupole 1; Q3, quadrupole 3; QQQ, triple quadrupole encompasses many conditions, ranging from atherosclerosis to myocardial infarction, and accounts for 30% of all deaths in developed and developing countries combined [1]. The incidence of this noncommunicable disease is expected to increase [2], placing a significant burden on the healthcare system and the economy. Although there are more than 150 putative CVD biomarker proteins in the literature [3–5], validation of these biomarkers requires the analysis of thousands of samples [6]. Likewise, the translation of these biomarkers into the clinic where they can be used for diagnosis and classification of different types of CVD also requires an accurate, robust, high-throughput assay. Many of these putative CVD biomarkers are plasma pro- teins, and plasma is already in common use for diagnos- tics and is the biofluid of choice for many clinical analyses. Plasma, however, is a highly complex matrix because it has a C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com