N‑Terminal\nProteomics Reveals Distinct Protein\nDegradation Patterns in Different Types of Human Atherosclerotic Plaques

Atherosclerotic plaque rupture is a major cause of cardiovascular\nevents. Plaque destabilization is associated with extracellular matrix\n(ECM) modification involving proteases which generate protein fragments\nwith new N-termini. We hypothesized that rupture-prone plaques would\ncontain elevated fragment levels, and their sequences would allow\nidentification of active proteases and target proteins. Plaques from\n21 patients who underwent surgery for symptomatic carotid artery stenosis\nwere examined in an observational/cross-sectional study. Plaques were\nanalyzed by liquid chromatography–mass spectrometry for the\npresence of N-terminal fragments. 33920 peptides were identified,\nwith 17814 being N-terminal species. 5735 distinct N-terminal peptides\nwere quantified and subjected to multidimensional scaling analysis\nand consensus clustering. These analyses indicated three clusters,\nwhich correlate with gross macroscopic plaque morphology (soft/mixed/hard),\nultrasound classification (echolucent/echogenic), and the presence\nof hemorrhage/ulceration. Differences in the fragment complements\nare consistent with plaque-type-dependent turnover and degradation\npathways. Identified peptides include signal and pro-peptides from\nsynthesis and those from protein fragmentation. Sequence analysis\nindicates that targeted proteins include ECM species and responsible\nproteases (meprins, cathepsins, matrix metalloproteinases, elastase,\nand kallikreins). This study provides a large data set of peptide\nfragments and proteases present in plaques of differing stability.\nThese species may have potential as biomarkers for improved atherosclerosis\nrisk profiling.