Proteome dissection of the atherosclerotic plaque: a deep immersion into the extracellular matrix ocean.

Background- Carotid plaque rupture, leading to atherothrombosis, is the third most common cause of death and it is responsible for 20% of all strokes. Plaque core-specific molecular factors of the extracellular matrix (ECM) can be identified by proteomics analysis and may be exploited as clinically relevant specific biomarkers of vulnerability. Aim of this study was to map ECM proteins and fragments from carotid plaque specimens. Methods-The ECM profile of atherosclerotic internal carotid artery (ICA) derived from endoarterectomy was obtained by using HPLC coupled with mass spectrometry (LC-MSMS). ICA samples were treated by different extraction methods and identified proteins in all fractions were evaluated and compared. All fractions were analysed by LC-MSMS prior and after fractionation by gel electrophoresis. Histology and immunohistochemistry were performed to characterize plaque core. Results-The LC-MSMS approach enabled the identification of more than 150 ECM and ECM associated proteins (according to Gene Ontology database). Among these the most represented families were proteoglycans and collagens. Gel electrophoresis fractionation evidenced peptides of Collagen alpha 1 and 2 (193 and 129 kDa respectively) at low MW regions (< 30 kDa) suggesting an intense intraplaque enzyme activity. Conclusions-An optimized workflow, allowing a detailed matrix protein profile of carotid plaque, may assist and improve biomarker discovery of molecular factors specifically related to disease complication. The proportion between intact protein and its fragments into the ECM plaque core could represent a quantitative indirect measure of intralesional protease activity and therefore provide a clinically novel and potentially more reliable index of “the chemical rupture risk” for vulnerable plaques.