Crosslinker design should be driven by data, not only concepts. Here, we argue that both concepts and functional groups need more thorough experimental evaluation, so that we can show exactly how and where they are useful when applied to crosslinkers. These groups are defined by concepts including: reaction selectivity to increase information density, enrichability to improve detection, cleavability to enhance the identification process and isotope-labelling for quantification. The anatomy of crosslinkers can be modular, sometimes comprising combinations of functional groups. The technology depends heavily on crosslinking reagents. Its appeal lies in a rapid workflow, high sensitivity and the ability to provide data on proteins in complex systems, even in whole cells. Crosslinking mass spectrometry has become a core technology in structural biology and is expanding its reach towards systems biology.
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