Comparative Analysis of Chemical Cross-Linking Mass Spectrometry Data Indicates That Protein STY Residues Rarely React with N-Hydroxysuccinimide Ester Cross-Linkers

When it comes to mass spectrometry data analysis foridentificationof peptide pairs linked by N-hydroxysuccinimide (NHS)ester cross-linkers, search engines bifurcate in their setting ofcross-linkable sites. Some restrict NHS ester cross-linkable sitesto lysine (K) and protein N-terminus, referred to as K only for short,whereas others additionally include serine (S), threonine (T), andtyrosine (Y) by default. Here, by setting amino acids with chemicallyinert side chains such as glycine (G), valine (V), and leucine (L)as cross-linkable sites, which serves as a negative control, we showthat software-identified STY-cross-links are only as reliable as GVL-cross-links.This is true across different NHS ester cross-linkers including DSS,DSSO, and DSBU, and across different search engines including MeroX,xiSearch, and pLink. Using a published data set originated from syntheticpeptides, we demonstrate that STY-cross-links indeed have a high falsediscovery rate. Further analysis revealed that depending on the dataand the search engine used to analyze the data, up to 65% of the STY-cross-linksidentified are actually K-K cross-links of the same peptidepairs, up to 61% are actually K-mono-links, and the rest tend to containshort peptides at high risk of false identification.