biostudies-literatureRuwolt MEuropean Research CouncilLeibniz-Forschungsinstitut f?r Molekulare Pharmakologie5248-5255https://www.ebi.ac.uk/biostudies/studies/S-EPMC10061366biostudies-literatureUnknown95(12)Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions <i>in vitro</i> and <i>in vivo</i>. 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>i.e.</i>, 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 founAnalytical chemistryReal-Time Library Search Increases Cross-Link Identification Depth across All Levels of Sample Complexity.PMC10061366949184He YBroichhagen JBarshop WViner RRuwolt MHuguet RBorges Lima DLiu FfalseReal-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 <i>in vitro</i> and <i>in vivo</i>. 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>i.e.</i>, 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 foun2023-01-01T00:00:00Z2023 Mar2026-05-29T07:50:10.24Z2024-11-21T05:10:07.978ZS-EPMC100613663692687210.1021/acs.analchem.2c05141