Proteomics characterization of extracellular space components in the human aorta.
Proteomics characterization of extracellular space components in the human aorta.
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DOI：
10.1074/mcp.m110.001693
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发表时间：
2010-09
期刊：
影响因子：
--
通讯作者：
Mayr M
中科院分区：
文献类型：
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作者：
Didangelos A;Yin X;Mandal K;Baumert M;Jahangiri M;Mayr M
血管细胞外基质（ECM）对于血管壁的结构完整性至关重要，并且还充当血管细胞分泌产物以及来自循环的分子结合和保留的基质。尽管蛋白质组学以前已应用于血管组织，但很少有研究专门针对血管 ECM 及其相关蛋白。因此，其详细组成仍有待表征。在这项研究中，我们描述了一种从人主动脉中提取细胞外蛋白并通过蛋白质组学进行鉴定的方法。该方法基于 (a) 有效脱细胞以富集稀缺的细胞外蛋白质，(b) ECM 蛋白质的成功溶解和去糖基化，以及 (c) 使用光谱计数相对估计蛋白质丰度。我们的三步提取方法鉴定出了 103 种细胞外蛋白，其中三分之一从未在血管组织的蛋白质组学文献中报道过。特别是，首次在人类主动脉的蛋白质水平上鉴定出三种糖蛋白（podocan、sclerostin 和 agrin）。我们还鉴定了细胞外脂肪细胞增强子结合蛋白 1、软骨糖蛋白阿孢菌素，以及先前假设的蛋白质，视网膜色素上皮 (RPE) spondin。此外，我们的方法使我们能够根据蛋白水解酶及其相应降解产物的鉴定来筛选主动脉样品中的蛋白水解。例如，我们能够通过质谱法检测基质金属蛋白酶 9，并将其存在与临床样本中纤连蛋白的降解联系起来。我们期望这种蛋白质组学方法能够进一步了解血管细胞外环境的组成，阐明 ECM 重塑和降解，并提供对重要病理过程的见解，例如斑块破裂、动脉瘤形成和再狭窄。
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.