Real-time library search assisted identification of cross-links increases resolution for structural modeling and interactome mapping

ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a universal tool of molecular and structural biology 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 because a large proportion of measurement time is spent on MS2 acquisitions of mono-links. 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/tert-butyl-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 full scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Furthermore, RTLS improves cross-link identification from unenriched samples and short gradients, which is beneficial in high-throughput approaches and when instrument time or sample amount is limited.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Suspension Culture

SUBMITTER: Max Ruwolt  

LAB HEAD: Fan Liu

PROVIDER: PXD037652 | Pride | 2023-03-20

REPOSITORIES: Pride