N‑Terminal Proteomics Reveals Distinct Protein Degradation Patterns in Different Types of Human Atherosclerotic Plaques

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