Myocardial ischemia biomarker discovery: multiple protein separation methodologies for analysis of albumin depleted and enriched fractions from serum

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Qin Fu1, Simon Sheng1, Rebekah Gundry1, Jia Zhu1, Miroslava Stastna1, Julie Miller1, Ola Forsstrom-Olsson2, Marjan Gucek1, Robert Cole1, Robert Cotter3, Eduardo Marban1, and Jennifer Van Eyk, jvaneyk1@jhmi.edu1. (1) Department of Medicine, Johns Hopkins University-Bayview Campus, 5200 Eastern Avenue, Mason F. Lord Building, Center Tower, Room 602, Baltimore, MD 21224, (2) Ludesi, Sweden, (3) Department of Pharmacology, Johns Hopkins University, Baltimore, MD
Ischemia markers for evaluating acute coronary syndrome (ACS) patients are essential for the early diagnosis and risk stratification of chest pain patients presenting to the emergency department. Our approach to biomarker discovery for myocardial ischemia centers on in-depth analysis of temporal serum samples obtained from patients undergoing cardiac catherization and/or angioplasty in which balloon inflation induces a timed ischemic event. In-depth proteomic analysis of serum samples obtained during the course of the evolving ischemia allows differentiation between potential ischemic biomarkers from traditional cellular necrosis markers (TnI). Serial serum samples were obtained under strict collection protocols and each patient's serum was partitioned into an HSA-enriched and immunoglobin/HSAdepleted fractions. The immunoglobulin/HAS depleted fractions were analyzed using 2DE and various LC techniques. Intact mass is determined by MALDI TOF MS for the differential LC fractions and used for validation of protein identification and post translational modifications. The detection of skeletal and cardiac muscle cellular proteins and cytokines indicated that the combined protein separation methods allow proteome coverage of 10e8. The protein composition of the serum changed over time in patients with evolving ischemia and was startling different from the healthy and disease controls.