Proteome-scale recombinant standards and a robust high-speed search engine to advance cross-linking ms-based interactomics

Advancing data analysis tools for proteome-wide cross-linking mass spectrometry (XL-MS) requires ground-truth standards that mimic biological complexity. Here, we develop wellcontrolled XL-MS standards comprising hundreds of recombinant proteins that are systematically mixed for cross-linking. We use one standard dataset to guide the development of Scout, a search engine for XL-MS with MS-cleavable cross-linkers. Using other, independent datasets from our standards as well as published datasets, we benchmark the performance of Scout and existing XL-MS software. This demonstrates that Scout offers the best combination of speed, sensitivity, and false-discovery rate control. These results illustrate how our large recombinant standards can support the development of XL-MS analysis tools and evaluation of XL-MS results. ### Competing Interest Statement FL is a shareholder and advisory board member of Absea Biotechnology Ltd., and an advisory board member of VantAI. TC is the co-founder of Absea Biotechnology Ltd. SW is an employee of Absea Biotechnology Ltd. The remaining authors have no competing interests.