Click-linking: a cell-compatible protein crosslinking method based on click chemistry

We developed a novel approach to in situ crosslinking mass spectrometry (XL-MS) where the reaction is split into two sequential and orthogonal coupling events. The method involves pre-stabilization of the proteome using a fixation protocol, followed by a two-step process that begins with extensive labeling of surface-available lysines with click-chemistry compatible reagents modified with NHS Esters. The second step involves the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of the installed precursors, which generates crosslinks with high efficiency. We observed approximately a 20-fold increase in protein-protein interactions detected by this approach when compared to standard DSS crosslinkers, and our Click-linking strategy even outperformed enrichable crosslinkers such as PhoX.