Rapid Mapping of Protein Interactions Using Tag‐Transfer Photocrosslinkers.

Analysing protein complexes by chemical crosslinking‐mass spectrometry (XL‐MS) is limited by the side‐chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue‐unbiased diazirine‐based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol‐containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this "tag and transfer" approach is demonstrated using a well‐defined peptide/protein regulatory interaction (BID80‐102/MCL‐1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA). Fast, enrichable photocrosslinking: Photocrosslinking reagents with methanethiosulfonate and diazirine functionalities have been developed. After crosslinking and reduction, a thiol tag stays at the interaction site, to be located by mass spectrometry and used for enrichment of even low abundance crosslinks. A UV LED irradiation platform reduces crosslinking times to 10 s. [ABSTRACT FROM AUTHOR]