Ynamide Coupling Reagent for the Chemical Cross-Linking of Proteins in Live Cells

Chemical
cross-linking of proteins coupled with mass
spectrometry
analysis (CXMS) is a powerful method for the study of protein structure
and protein–protein interactions (PPIs). However, the chemical
probes used in the CXMS are limited to bidentate reactive warheads,
and the available zero-length cross-linkers are restricted to 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS)
and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMTMM). To alleviate this issue, an efficient coupling reagent, sulfonyl
ynamide, was developed as a new zero-length cross-linker that can
connect high-abundance carboxyl residues (D/E) with lysine (K) to
form amide bonds in the absence of any catalyst. Significant improvement
in the cross-linking efficiency and specificity in comparison with
traditional EDC/NHS was achieved with model proteins, which includes
inter- and intramolecular conjugations. The cross-linked structures
were validated by X-ray crystallography. Importantly, this coupling
reagent can be successfully used to capture interacting proteins in
the whole proteome and can be a useful reagent for probing potential
protein–protein interactions in situ.