In vivo Cross-Linking Mass Spectrometry for Protein and Complex Structural Analysis

Chemical cross-linking of proteins with mass spectrometry (XL-MS) has increasingly become a powerful technique when studying protein structures and complexes. This approach is based on the reactivity of cross-linkers to specific protein sites - usually primary amines, including side chains of lysine residues and protein N-termini which yields information on protein-protein interactions and protein conformations. Information provided by XL-MS is complementary to that from other structural methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. Here, we describe a protocol for in-house synthesis and use of a peptide-based cross-linker with optimized features for interactome studies of complex biological samples. These features comprise the protein interaction reporter (PIR) technology, MS-cleavable bonds, and an affinity tag, which ultimately facilitate the identification of cross-linked peptide pairs. The membrane permeability enables the cross-linking of living cells, tissues, and isolated organelles (e.g., nuclei and mitochondria), providing valuable structural and interaction data on proteins as they exist in their native environment. Moreover, quantitative XL-MS can be utilized for comparative interactome studies, providing information on protein conformational and interaction changes between varying biological states.