Yugandhar, K. et al. Mol. Cell. Proteomics https://doi.org/10.1074/mcp.TIR119.001847 (2019).
Determining the 3D structure of proteins and the structural basis of protein–protein interactions requires determining the spatial constraints between interacting partners. One method to capture these interactions is cross-linking mass spectrometry (XL-MS), and efficient MS-cleavable chemical cross-linkers have allowed the approach to be expanded to the proteome scale. Yugandhar et al. have developed MaxLinker, an MS3-centric cross-link search approach that the authors demonstrate to have a significantly lower misidentification rate than the standard MS2-only approach. MaxLinker starts with MS3-level cross-link candidates and discards the ones without reliable sequence information for at least one of the two cross-linked peptides. This is followed by an MS2-based rescue step that looks at discarded peptides that may have partial sequence information at this level. The authors demonstrate the search strategy on a human proteome-wide XL-MS experiment using K562 cells. More than 9,000 unique cross-links were identified at a 1% false discovery rate. The software is freely available for download from the lab website.
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Singh, A. MS3-based cross-link search platform. Nat Methods 17, 129 (2020). https://doi.org/10.1038/s41592-020-0752-9