Extracellular matrix characterization in plaques from carotid endarterectomy by a proteomics approach

Abstract

Extracellular matrix (ECM) is a fundamental component of multicellular organisms. Alteration of its structure and/or molecular composition are associated with several pathologies, among the others with atherosclerosis. To determine how the overall ECM architecture of a tissue as complex as the atheroma may change under varying pathological conditions constitutes a great technical challenge. This entails removing cell components and solubilisation of fibrillar proteins in order to allow enzymatic digestion and mass spectrometry analyses. This work aimed at testing and assessing an easy to use, standardized and reproducible proteomics protocol to map ECM proteins in carotid plaque specimens. To this end, plaques from endarterectomies were incubated in different buffers and the resulting solutions and tissue homogenates after incubation were processed for mass spectrometry. Comparison of the different workflows showed that 4M Guanidine treatment (following 0.5M NaCl and 0.08% SDS incubations) is the most promising in terms of ECM enrichment. The protocol can also be used to identify different and complementary classes of proteins both in plaque extracts and homogenates.

Keywords: Atherosclerotic plaque; Carotid endarterectomy; Extracellular matrix proteins; Proteomics analysis.