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.