P5721Multiplexed measurement of candidate protein biomarkers of cardiovascular disease in blood

Abstract Background/Introduction Almost half of all deaths in Europe are attributable to Cardiovascular Disease (CVD). There is a critical unmet need for better biomarkers so that CVD can be diagnosed at an earlier stage and with greater accuracy. Mass spectrometry-based multiple reaction monitoring (MRM) allows for rapid, targeted measurement of multiple protein biomarkers via detection of “proteotypic” peptide fragments. MRM assays can be developed in blood samples, which is desirable for minimally-invasive, routine monitoring of cardiovascular health. Purpose The purpose of this study was to design a robust MRM method for the simultaneous detection and measurement of a panel of 36 proteins, including 19 known and 17 novel protein biomarkers of CVD, in blood. The overall aim was to evaluate the potential clinical utility of this biomarker panel for prediction of heart failure in a cohort of 500 patients. Methods Proteotypic peptides for each of the 36 candidate biomarker proteins were identified using Skyline and Spectrum Mill PeptideSelector software. MRM assays were compiled into a single analytical method that was run on a 6460 triple quadrupole mass spectrometer, utilizing nanoflow reverse phase C18 chromatographic ChipCube based separation. Patient samples and synthetic peptides were used to obtain reference MS/MS spectra for building high quality MRM assays. For initial evaluation of the candidate protein biomarker panel, the MRM method was applied, in a sample blinded manner, to a cohort of 500 serum samples from patients with heart failure and age-matched non-heart failure controls with CVD risk-factors. Results MRM assays were established for 36 peptides, corresponding to 9 of the known candidate biomarker proteins and all 17 of the novel biomarkers. Individually, a number of the biomarker proteins show differential expression between heart failure with preserved and reduced ejection fraction. Combined measurement of all biomarker proteins was found to have greater predictive capacity for heart failure than the current gold standard of B-type natriuretic peptide (BNP) alone. A statistical model found that the biomarker panel was capable of correctly predicting heart failure in blinded patient samples (50 heart failure vs 50 non-heart failure) with 74% sensitivity and 64% specificity. Conclusion(s) Through this study, an MRM method been developed for robust, multiplexed measurement of 26 CVD biomarker proteins in patient serum samples. Here it wa