Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization
Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization
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DOI：
10.1021/acs.jproteome.7b00527
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发表时间：
2017-11-01
影响因子：
4.4
通讯作者：
Hansen, Kirk C.
中科院分区：
文献类型：
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作者：
Barrett, Alexander S.;Wither, Matthew J.;Hansen, Kirk C.
细胞外基质（ECM）很容易通过用非变性洗涤剂去细胞化组织来富集，以溶解和消耗绝大多数细胞组分。这种方法已被广泛用于生成再生医学技术的ECM支架，并在3D细胞培养中模拟ECM如何促进疾病进展。然后可以使用强离液剂缓冲液产生高度富集的ECM级分，所述强离液剂缓冲液与下游自下而上蛋白质组学分析或广泛透析后的3D细胞培养实验相容。对于大多数组织，在离液剂提取后保留不溶性沉淀，其富含结构ECM组分。以前，我们表明，这一未充分研究的部分代表约80%的总纤维胶原蛋白从肺和其他ECM纤维成分，是已知的共价交联。在这里，我们提出了一种羟胺消化的方法，与比较，已建立的CNBr方法的基质体表征的离液液不溶性ECM分析。由于ECM特征在组织之间差异很大，我们选择了五种代表独特和不同ECM丰度、组成和生物力学特性的组织。羟胺消化与下游蛋白质组学工作流程相容，从不溶性ECM级分产生高水平的ECM肽，并且与CNBr消化相比降低了分析变异性。数据可通过ProteomeXchange获得，标识符为PXD 006428。
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.