Lima, D. B. | bioRxiv Science

bioRxiv2023

PROTEOME-SCALE RECOMBINANT STANDARDS AND A ROBUST HIGH-SPEED SEARCH ENGINE TO ADVANCE CROSS-LINKING MS-BASED INTERACTOMICS

AO_SCPLOWBSTRACTC_SCPLOWAdvancing 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.

bioinformatics↗

bioRxiv2022

Real-time library search increases cross-link identification depth across all levels of sample complexity

Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

systems biology↗