Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States.
Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, Wisconsin 53705, United States.
Anal Chem. 2020 Dec 15;92(24):15693-15698. doi: 10.1021/acs.analchem.0c03104. Epub 2020 Nov 24.
The extracellular matrix (ECM) provides an architectural meshwork that surrounds and supports cells. The dysregulation of heavily post-translationally modified ECM proteins directly contributes to various diseases. Mass spectrometry (MS)-based proteomics is an ideal tool to identify ECM proteins and characterize their post-translational modifications, but ECM proteomics remains challenging owing to the extremely low solubility of the ECM. Herein, enabled by effective solubilization of ECM proteins using our recently developed photocleavable surfactant, Azo, we have developed a streamlined ECM proteomic strategy that allows fast tissue decellularization, efficient extraction and enrichment of ECM proteins, and rapid digestion prior to reversed-phase liquid chromatography (RPLC)-MS analysis. A total of 173 and 225 unique ECM proteins from mouse mammary tumors have been identified using 1D and 2D RPLC-MS/MS, respectively. Moreover, 87 (from 1DLC-MS/MS) and 229 (from 2DLC-MS/MS) post-translational modifications of ECM proteins, including glycosylation, phosphorylation, and hydroxylation, were identified and localized. This Azo-enabled ECM proteomics strategy will streamline the analysis of ECM proteins and promote the study of ECM biology.
细胞外基质 (ECM) 提供了一个围绕和支持细胞的建筑网格。高度翻译后修饰的 ECM 蛋白的失调直接导致各种疾病。基于质谱 (MS) 的蛋白质组学是识别 ECM 蛋白并表征其翻译后修饰的理想工具，但由于 ECM 极低的溶解度，ECM 蛋白质组学仍然具有挑战性。在此，我们利用最近开发的光裂解表面活性剂 Azo 有效地溶解 ECM 蛋白，开发了一种简化的 ECM 蛋白质组学策略，该策略允许快速组织脱细胞化、高效提取和富集 ECM 蛋白，以及在反相液相色谱 (RPLC)-MS 分析之前快速消化。使用 1D 和 2D RPLC-MS/MS 分别从小鼠乳腺肿瘤中鉴定出 173 和 225 种独特的 ECM 蛋白。此外，鉴定和定位了 87 种（来自 1DLC-MS/MS）和 229 种（来自 2DLC-MS/MS）ECM 蛋白的翻译后修饰，包括糖基化、磷酸化和羟化。这种 Azo 增强的 ECM 蛋白质组学策略将简化 ECM 蛋白的分析，并促进 ECM 生物学的研究。
