We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, post-translationally modified (PTM) via N- and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra- and intermolecular cross-linking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications.

我们报告了一种多重成像质谱方法，该方法可在福尔马林固定石蜡包埋的组织切片上空间定位并选择性地访问细胞外基质。细胞外基质 (ECM) 由 (1) 通过 N- 和 O- 连接糖基化以及脯氨酸和赖氨酸羟基化进行翻译后修饰 (PTM) 的纤维蛋白和 (2) 糖胺聚糖修饰的蛋白聚糖组成。访问所有这些组件提出了独特的分析挑战。由于 ECM 肽丰度低、分子内和分子间交联以及 PTM 较低，通过胰蛋白酶进行的传统肽分析无法有效捕获 ECM 肽。在之前的研究中，我们开发了基质辅助激光解吸电离成像质谱 (MALDI-IMS) 技术，通过 III 型胶原酶消化捕获胶原蛋白肽，无论是单独消化还是通过 PNGaseF 消化去除 N-聚糖后捕获胶原蛋白肽。然而，在纤维化组织中，除胶原型蛋白之外的 ECM 成分（包括弹性蛋白和糖胺聚糖）的积累，限制了任何单一酶接触复杂 ECM 的功效。在这里，我们开发了一种新颖的系列酶策略，用于从单个组织切片中定义细胞外基质，包括 PTM，用于 MALDI-IMS 应用。