Department of Chemistry and Biochemistry, University of Texas, Arlington, TX, USA.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
Methods Mol Biol. 2023;2690:255-267. doi: 10.1007/978-1-0716-3327-4_22.
Protein-protein interactions (PPIs) are the physical interactions formed among proteins. These interactions are primarily functional, i.e., they arise from specific biomolecular events, and each interaction interface serves a specific purpose. A significant number of methods have been developed for protein interactions in the field of proteomics in the last decade. Advanced mass spectrometry technology significantly contributed to the development of these methods. The rapid advancement of groundbreaking MS technology has greatly aided the mapping of protein interaction from large-data sets comprehensively. This chapter describes the affinity purification (AP) mass spectrometry (MS)-based methods combined with chemical cross-linking (XL) of protein complexes. This chapter includes sample preparation methods involving cell culture, cell treatments with ligands, drugs, and cross-linkers, protein extractions, affinity purification, sodium dodecyl sulfate (SDS) polyacrylamide gel separation, in-solution or in-gel digestion, liquid-chromatography, and mass spectrometry analysis of samples (LC-MS/MS). Application of a cleavable cross-linker, dual cleavable cross-linking technology (DUCCT) in combination with the affinity purification (AP) method has also been described. Methods for data analysis using unmodified and cross-linked peptide analysis are discussed.
蛋白质-蛋白质相互作用（PPIs）是蛋白质之间形成的物理相互作用。这些相互作用主要是功能性的，即它们源于特定的生物分子事件，每个相互作用界面都有特定的用途。在过去十年的蛋白质组学领域，已经开发出了许多用于蛋白质相互作用的方法。先进的质谱技术为这些方法的发展做出了重大贡献。突破性 MS 技术的快速发展极大地促进了从大数据集全面映射蛋白质相互作用。本章描述了基于亲和纯化（AP）的质谱（MS）与蛋白质复合物的化学交联（XL）相结合的方法。本章包括涉及细胞培养、用配体、药物和交联剂处理细胞、蛋白质提取、亲和纯化、十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶分离、溶液内或胶内消化、液相色谱和样品的质谱分析（LC-MS/MS）的样品制备方法。还描述了在亲和纯化（AP）方法中结合可裂解交联剂、双可裂解交联技术（DUCCT）的应用。讨论了使用未修饰和交联肽分析进行数据分析的方法。
