Proteomic Profiling of Acute Cardiac Allograft Rejection

Abstract

Proteome analysis has emerged as a valuable tool for the study of large-scale protein expression profiles. Here, we applied this novel technology to identify specific biomarkers for acute cardiac allograft rejection. Hearts of C57BL/10 mice were placed in fully major histocompatibility complex-mismatched C3H/He recipients. Syngeneic transplants served as controls. Intragraft protein expression analysis was performed using fluorescence two-dimensional difference gel electrophoresis on day 6 posttransplant. Spots of interest were subsequently subjected to nanospray ionization tandem mass spectrometry (MS/MS) for protein identification. In addition, expression of selected proteins was confirmed by Western blot analysis and by immunohistochemistry. Two-dimensional difference gel electrophoresis enabled detection of 1541 protein spots. For 95 protein spots, the expression level during acute rejection differed by more than 1.5-fold from that observed in syngeneic grafts. Spots with significant differential regulation identified by tandem mass spectrometry were derived from peroxiredoxin 6, pyruvate kinase isozyme M2, coronin 1A, protein disulfide isomerase A3 precursor, and aconitate hydratase. These identified proteins may constitute novel biomarkers of acute cardiac allograft rejection and might hold great potential as surrogate markers for monitoring in vivo alloimmune response.