Abstract
We developed a pipeline to integrate the proteomic technologies used from the discovery to the verification stages of plasma biomarker identification and applied it to identify early biomarkers of cardiac injury from the blood of patients undergoing a therapeutic, planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy. Sampling of blood directly from patient hearts before, during and after controlled myocardial injury ensured enrichment for candidate biomarkers and allowed patients to serve as their own biological controls. LC-MS/MS analyses detected 121 highly differentially expressed proteins, including previously credentialed markers of cardiovascular disease and >100 novel candidate biomarkers for myocardial infarction (MI). Accurate inclusion mass screening (AIMS) qualified a subset of the candidates based on highly specific, targeted detection in peripheral plasma, including some markers unlikely to have been identified without this step. Analyses of peripheral plasma from controls and patients with PMI or spontaneous MI by quantitative multiple reaction monitoring mass spectrometry or immunoassays suggest that the candidate biomarkers may be specific to MI. This study demonstrates that modern proteomic technologies, when coherently integrated, can yield novel cardiovascular biomarkers meriting further evaluation in large, heterogeneous cohorts.
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Acknowledgements
The authors gratefully acknowledge support from the US National Institutes of Health (NIH) National Heart, Lung, and Blood Institute U01HL083141 and R01HL096738-02 to R.E.G., S.A.C. and M.S.S., the Donald W. Reynolds Foundation (to R.E.G. and M.S.S.) and Foundation Leducq (to R.E.G.). S.A.C. also acknowledges support from the NIH 1U24 CA126476 as part of the National Cancer Institute (NCI)'s Clinical Proteomic Technologies Assessment in Cancer Program, from the Women's Cancer Research Fund of the Entertainment Industry Foundation and to D.R.M. from the NCI Clinical Proteomic Technologies Initiative (R01 CA126219). We also appreciate the excellent technical assistance of C. Bodycombe.
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T.A.A., S.A.C. and R.E.G. wrote the manuscript. S.A.C. and M.A.G. conceived of the biomarker pipeline used here. R.E.G. conceived of the PMI as a model for proteomic discovery, and along with M.A.F., M.S.S., G.D.L. and L.A.F. developed the human studies protocols included in the manuscript, and performed the phenotyping of the patient populations. H.K., X.S. and T.A.A. carried out all of the sample preparation, conducted the MS-based proteomics experiments for discovery and AIMS and interpreted the results. H.K. and M.B. conducted all of the MRM-MS experiments for assaying proteins by M.S.S. X.S. tested, developed and applied all antibody-based measurements, with contributions from D.S. M.A.G. was responsible for design of the AIMS experiments. K.R.C. designed and adapted the Spectrum Mill software for peptide and protein identification, label-free quantification and calculation of peptide-level FDR and participated in data analysis. D.R.M., M.S.S. and K.R.C. were responsible for statistical design and analysis.
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Supplementary Tables 1–9, Supplementary Figs. 1–6, Supplementary Results and Discussion, and Supplementary Methods (PDF 11166 kb)
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Addona, T., Shi, X., Keshishian, H. et al. A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease. Nat Biotechnol 29, 635–643 (2011). https://doi.org/10.1038/nbt.1899
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DOI: https://doi.org/10.1038/nbt.1899
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