Abstract
The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.
Highlights
The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation
We identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin
We were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis
Summary
Athanasios Didangelos‡, Xiaoke Yin‡, Kaushik Mandal§, Mark Baumert¶, Marjan Jahangiri㥋, and Manuel Mayr‡**. The vascular ECM serves as a substrate for the binding and retention of secreted, soluble proteins of vascular cells as well as molecules coming from the circulation, including lipoproteins, growth factors, cytokines, proteases, and protease inhibitors These components are invariably associated with ECM proteins, especially proteoglycans. The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; GAG, glycosaminoglycan; Bis-Tris, 2-[bis(2hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ANOVA, analysis of variance; AEBP1, adipocyte enhancer-binding protein 1; RPE, retinal pigment epithelium; LTQ, linear trap quadrupole. MS); and third, to interface the nanoflow LC system to a recently developed injection device, which splits the flow from the analytical column, to allow the reanalysis of the same sample during a single LC-MS/MS run (RePlay, Advion)
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