Xlink-Identifier: An Automated Data Analysis Platform for Confident Identifications of Chemically Cross-Linked Peptides Using Tandem Mass Spectrometry

Chemical cross-linking combined with mass spectrometry provides a powerful method for identifying protein−protein interactions and probing the structure of protein complexes. A number of strategies have been reported that take advantage of the high sensitivity and high resolution of modern mass spectrometers. Approaches typically include synthesis of novel cross-linking compounds, and/or isotopic labeling of the cross-linking reagent and/or protein, and label-free methods. We report Xlink-Identifier, a comprehensive data analysis platform that has been developed to support label-free analyses. It can identify interpeptide, intrapeptide, and deadend cross-links as well as underivatized peptides. The software streamlines data preprocessing, peptide scoring, and visualization and provides an overall data analysis strategy for studying protein−protein interactions and protein structure using mass spectrometry. The software has been evaluated using a custom synthesized cross-linking reagent that features an enrichment tag. Xlink-Identifier offers the potential to perform large-scale identifications of protein−protein interactions using tandem mass spectrometry.

化学交联（chemical cross-linking）结合质谱（mass spectrometry）技术，是用于鉴定蛋白质-蛋白质相互作用（protein−protein interactions）、探究蛋白质复合物（protein complexes）结构的强有力研究手段。目前已有诸多研究策略依托现代质谱仪（mass spectrometers）的高灵敏度与高分辨率得以开发。这类策略通常涵盖新型交联化合物（cross-linking compounds）的合成、交联试剂与/或蛋白质的同位素标记（isotopic labeling），以及无标记方法（label-free methods）三类。本研究报道了一款名为Xlink-Identifier的综合性数据分析平台，其专为支持无标记分析而开发。该平台可鉴定肽间交联（interpeptide cross-links）、肽内交联（intrapeptide cross-links）、死端交联（deadend cross-links）以及未衍生化肽段（underivatized peptides）。这款软件可优化数据预处理（data preprocessing）、肽段评分（peptide scoring）与可视化（visualization）流程，并为依托质谱技术开展蛋白质-蛋白质相互作用及蛋白质结构研究提供了一套完整的数据分析方案。本研究通过一款带有富集标签的定制合成交联试剂对该软件进行了性能验证。Xlink-Identifier具备利用串联质谱（tandem mass spectrometry）实现大规模蛋白质-蛋白质相互作用鉴定的应用潜力。