N-Terminal proteomics reveals distinct protein degradation patterns in different types of human atherosclerotic plaques

Abstract

BACKGROUNDDestabilization and rupture of atherosclerotic plaques is a major cause of acute atherosclerotic cardiovascular events, including heart attack, ischemic stroke and peripheral arterial disease. Plaque destabilization is associated with extracellular matrix (ECM) modification and remodelling involving protease activity. Enzymatic cleavage generates protein fragments with new ends (N-termini). We hypothesized that plaques susceptible to rupture would contain elevated levels of fragmented proteins with new N-termini. Identification of active proteases and their target proteins might allow categorization of plaque stability. METHODSPlaques from 21 patients who underwent carotid surgery due to symptomatic carotid artery stenosis were examined in an observational/cross-sectional study. The plaques were solubilized, digested, enriched for N-terminal fragments and analyzed by liquid chromatography-mass spectrometry. RESULTSThe above methodology detected 35349 peptides, with 19543 being N-terminal species; 6561 were subsequently identified and quantified. Multidimensional scaling analysis and hierarchical clustering indicate the presence of three distinct clusters, which correlate with gross macroscopic plaque morphology (soft, mixed, and hard), ultrasound classification (echolucent/echogenic) and presence of hemorrhage/ulceration. Major differences were identified in the complement of peptide fragments, consistent with alternative turnover and degradation pathways dependent on plaque type. Identified peptides include signal and pro-peptides from ECM synthesis/turnover, and many from protein fragmentation. Sequence analysis indicates the targeted proteins (including ECM species) and the proteases (including meprins, cathepsins, matrix metalloproteinases, elastase, kallikreins) involved in fragment generation. CONCLUSIONSThis study provides a large dataset of peptide fragments and proteases involved in plaque stability, mechanistic insights into remodelling, and possible biomarkers for improved atherosclerosis risk profiling. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/594251v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@f5e62forg.highwire.dtl.DTLVardef@1db3b07org.highwire.dtl.DTLVardef@7fe3org.highwire.dtl.DTLVardef@cc1329_HPS_FORMAT_FIGEXP M_FIG C_FIG