Signal Attrition in Whole Cell Crosslinking Mass Spectrometry

We investigated points in the typical sample workflow that could contribute to the perceived low cross-linking yields. We demonstrate, through a 2-step cross-linking strategy called Click-Linking, that the chemical reaction is not the main contributor to the low number of identified cross-links. We also demonstrate that sample preparation, digestion and peptide recovery are not causing biased losses against crosslinks. The low detection rates come mostly from severe crosslink signal splitting, as residues are participating in complex networks of reactive sites.