Optimizing TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies

TMT-based quantitative proteomics is one of the most efficient and reliable methods in relative protein quantification and has made indispensable contributions in protein biology. To enable quantitative interactomic studies for protein-protein interaction profiling, we hereby developed a TMT-based quantitative cross-linking mass spectrometry workflow based on gas-phase cleavable cross-linker DSSO on a FAIMS enabled Obitrap Fusion Lumos mass spectrometer. For benchmarking, we tested different HCD fragmentation energies and acquisition strategies on TMT-labeled cross-links. We also constructed a two interactome dataset by preparing cross-linked E. coli and HeLa lysates and mixed them with different ratios to assess the level of ion interference. Collectively, we demonstrated a stepHCD method with optimized energies delivers the best result, as demonstrated by the highest number of identified and quantified cross-links as well as the most accurate quantitative information.