A Membrane‐Permeable and Immobilized Metal Affinity Chromatography (IMAC) Enrichable Cross‐Linking Reagent to Advance In Vivo Cross‐Linking Mass Spectrometry

Cross‐linking mass spectrometry (XL‐MS) is an attractive method for the proteome‐wide characterization of protein structures and interactions. Currently, the depth of in vivo XL‐MS studies is lagging behind the established applications to cell lysates, because cross‐linking reagents that can penetrate intact cells and strategies to enrich cross‐linked peptides lack efficiency. To tackle these limitations, we have developed a phosphonate‐containing cross‐linker, tBu‐PhoX, that efficiently permeates various biological membranes and can be robustly enriched using routine immobilized metal ion affinity chromatography. We have established a tBu‐PhoX‐based in vivo XL‐MS approach that enables cross‐links in intact human cells to be identified in high numbers with substantially reduced analysis time. Collectively, the developed cross‐linker and XL‐MS approach pave the way for the comprehensive XL‐MS characterization of living systems. A phosphonate‐containing cross‐linker, tBu‐PhoX, has been developed that efficiently permeates various biological membranes and can be robustly enriched using routine immobilized metal ion affinity chromatography (IMAC). The optimized tBu‐PhoX‐based in vivo cross‐linking mass spectrometry (XL‐MS) system enables the time‐efficient and detailed characterization of protein interaction landscapes in intact human cells.