Chemical crosslinking coupled to high-resolution mass spectrometry (XL-MS) provides spatial restraints that resolve the architecture and dynamics of protein complexes beyond the limits of crystallography or cryo-EM. Yet, comprehensive crosslink detection remains limited by low reaction yields, incomplete enrichment, and the sensitivity of peptide detection. Here, we describe optimisation of Sulfo-SDA-based XL-MS on Thermo Orbitrap Eclipse and Orbitrap Astral instruments, benchmarked using recombinant proteins and immunoprecipitated native protein complexes. Sulfo-SDA combines an NHS ester with a photo-reactive diazirine, enabling broad amino-acid reactivity and temporal control of crosslink formation. We refined crosslinking conditions to enhance yield while maintaining structural integrity of protein assemblies. Empirical optimisation of UV activation, protein-to-linker ratios, and detergent compatibility improved recovery of inter- and intra-protein crosslinks from both purified and IP-derived material.
Crosslinked peptides were analysed using complementary fragmentation and enrichment workflows, that also incorporated FAIMS.Sensitive and accurate identification of crosslinked peptides was highly dependent on optimising chromatographic performance, particularly through appropriate pairing of column capacity with gradient length. For identification, spectra were processed using XiSearch, which supports non-cleavable and photo-activated chemistries such as Sulfo-SDA. XiSearch enabled high-confidence assignment of crosslinked peptide pairs through dedicated scoring of fragment ion series, probabilistic site localisation, and modelling of diazirine-derived mass shifts. Its flexible search space handling and false-discovery rate (FDR) control were critical for distinguishing true crosslinks from the expanded product mixtures characteristic of photo-crosslinking.Together, these optimisations establish a robust XL-MS workflow applicable to both recombinant and immunoprecipitated protein complexes. By integrating refined Sulfo-SDA chemistry with the high-throughput capability of the Orbitrap Astral platform, we achieve deeper structural coverage and improved quantitative reproducibility, paving the way for routine application of XL-MS in dynamic interactome mapping and structural proteomics.