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

Crosslinking mass spectrometry (XL-MS) has the potential to map the human interactome at high resolution and with high fidelity, replacing indirect, error prone sampling methods such as affinity pulldown MS. However, the sampling depth of XL-MS remains stubbornly low. We present a novel crosslinking strategy that splits the crosslinking reaction into two sequential and orthogonal coupling events. The method involves pre-stabilizing the spatial proteome with a fixation protocol inspired by immunofluorescence imaging, followed by a stepwise process that begins with extensively labeling surface-accessible lysines in the cell with N-hydroxysuccinimide (NHS)-modified click reagents. We show that a subsequent copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of the installed precursors generates crosslinks at levels approaching 30% of the total signal, as demonstrated by a subtractive approach. The method generates no detectable side reactions or obvious distortions of the spatial proteome. Protein-protein interactions (PPIs) are detected at levels approximately 20 times higher than a conventional DSS-based method, outperforming even enrichable crosslinkers.