Protein complexes within cells are fundamental to biological understanding, yet cataloging protein-protein interactions at the proteome level remains a significant challenge due to technical limitations of standard methodologies. This study evaluates the efficacy of crosslinking mass spectrometry (XL-MS) for large-scale identification of protein-protein interactions within Toxoplasma gondii. Utilizing the soluble fraction of tachyzoite lysates, we used a novel, MS-cleavable, and click-enrichable crosslinker, azide-A-DSBSO, alongside mass spectrometry optimization techniques including stepped-HCD coupled with FAIMS. This approach significantly enhanced the detection of crosslinks, yielding over 11,000 crosslink spectra, 3,000 unique residue-to-residue crosslinks, and 300 protein-protein interactions. Remarkably, the majority of these interactions corroborate with previously established hyperLOPIT data. Further analysis focused on the GRA1 protein, revealing a detailed interactome through immunoprecipitation combined with XL-MS. Crosslink mapping to existing crystal, cryo-EM, and AlphaFold structures confirmed that the distances between crosslinks predominantly align with theoretical expectations. Our findings underscore the utility of XL-MS as a powerful tool for elucidating the proteome-level interactome and extracting structural insights into proteins. This technique holds promise for investigating complex and elusive structures, such as cyst walls and dense granules, and for comprehensive interactome cataloging through the integration of hyperLOPIT and XL-MS.

[Funding: NIHAI134753]