In vivo cross-linking mass spectrometry: Advances and challenges in decoding protein conformational dynamics and complex regulatory networks in living cells

In vivo chemical cross-linking mass spectrometry (XL-MS) has emerged as a powerful technique for high-throughput, proteome-wide mapping of intramolecular conformations and intermolecular interactions of protein complexes in living cells. By providing distance constraints between specific residues, XL-MS enables the characterization of protein conformations and interaction networks under near-physiological conditions, greatly facilitating the analysis of biomacromolecular functions and regulatory mechanisms. The information obtained from cross-linking is particularly valuable at the systems level, and its value continues to increase with improvements in the density of cross-link identification, the precision of distance constraints, and the spatiotemporal resolution. In recent years, advances in cross-linker design, cross-linked peptide enrichment methods, mass spectrometry analysis, and artificial intelligence-assisted data analysis have significantly expanded the capabilities of in vivo XL-MS. This article systematically reviews the latest progress in in vivo XL-MS for protein conformation and interaction network analysis, highlights its unique advantages, discusses current technical challenges, and explores further development.