Abstract
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).
Highlights
Analysing protein complexes by chemical crosslinking-mass spectrometry (XL-MS) is limited by the sidechain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes
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)
XL-MS methods have been enhanced by new data analysis software,[2]
Summary
OmpA, respectively.[18] MTS-diazirine or MTS-TFMD conjugation of both variants was efficient (Figure S11), and did not prevent folding (Figure S12). The Skp/OmpA complex was assembled by dilution of urea-denatured OmpA into a Skp-containing solution, and PI-. The crosslinked Skp/OmpA complex was detected by non-reducing SDS-
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