Department of Biochemistry and Molecular Genetics, and ‡Biological Mass Spectrometry Facility, University of Colorado Denver , Aurora, Colorado 80045, United States.
J Proteome Res. 2017 Nov 3;16(11):4177-4184. doi: 10.1021/acs.jproteome.7b00527.
The extracellular matrix (ECM) is readily enriched by decellularizing tissues with nondenaturing detergents to solubilize and deplete the vast majority of cellular components. This approach has been used extensively to generate ECM scaffolds for regenerative medicine technologies and in 3D cell culture to model how the ECM contributes to disease progression. A highly enriched ECM fraction can then be generated using a strong chaotrope buffer that is compatible with downstream bottom-up proteomic analysis or 3D cell culture experiments after extensive dialysis. With most tissues, an insoluble pellet remains after chaotrope extraction that is rich in structural ECM components. Previously, we showed that this understudied fraction represented approximately 80% of total fibrillar collagen from the lung and other ECM fiber components that are known to be covalently cross-linked. Here, we present a hydroxylamine digestion approach for chaotrope-insoluble ECM analysis with comparison to an established CNBr method for matrisome characterization. Because ECM characteristics vary widely among tissues, we chose five tissues that represent unique and diverse ECM abundances, composition, and biomechanical properties. Hydroxylamine digestion is compatible with downstream proteomic workflows, yields high levels of ECM peptides from the insoluble ECM fraction, and reduces analytical variability when compared to CNBr digestion. Data are available via ProteomeXchange with identifier PXD006428.
细胞外基质（ECM）可通过非变性去污剂脱细胞化来丰富，从而溶解并耗尽绝大多数细胞成分。这种方法已被广泛用于生成用于再生医学技术的 ECM 支架，并用于 3D 细胞培养以模拟 ECM 如何促进疾病进展。然后，可以使用强变溶剂缓冲液生成高度富集的 ECM 级分，该缓冲液与下游自下而上的蛋白质组分析或 3D 细胞培养实验兼容，在经过广泛的透析后使用。对于大多数组织，在变溶剂提取后会留下富含结构性 ECM 成分的不可溶性沉淀。以前，我们表明，这一研究较少的部分代表了肺和其他已知共价交联的 ECM 纤维成分的总纤维胶原的约 80%。在这里，我们提出了一种用于变溶剂不可溶 ECM 分析的羟胺消化方法，并与用于基质组学表征的已建立的 CNBr 方法进行了比较。由于 ECM 特性在组织之间差异很大，我们选择了五种组织，这些组织代表了独特和多样化的 ECM 丰度、组成和生物力学特性。羟胺消化与下游蛋白质组学工作流程兼容，可从不可溶 ECM 级分中产生高水平的 ECM 肽，与 CNBr 消化相比，可降低分析变异性。数据可通过 ProteomeXchange 以标识符 PXD006428 获得。
