Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS, aka XL-MS) has become an established method for probing the conformation of a protein and for mapping the interface of protein-protein interactions. Central to this technology are chemical cross-linkers. The most popular cross-linkers such as DSS, BS3, and DSSO are all NHS esters, which react with protein amino groups relatively slowly over 10-60 minutes while in competition with the hydrolysis reaction of NHS esters. Concerned with the imperfections of NHS ester cross-linkers, we developed a new class of amine-selective nonhydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA cross-linking of proteins takes only 10 seconds under near physiological conditions. Besides, DOPA works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride (GdnHCl), making it possible to capture conformational changes of proteins during the unfolding or refolding process over a time scale of minutes.
The test results on six model proteins showed that 80% or more of the DOPA cross-links are compatible with protein crystal structures. Using two heterodimeric complexes of weakly interacting subunits, we demonstrated that DOPA2 (spacer arm 17.5 Å, maximal C-C distance 30.2 Å) fixed weak or transient protein-protein interactions better than DSS. We also demonstrated that DOPA2 indeed captured the structural change associated with the unfolding of ribonuclease A induced by either 8 M urea or 6 M GdnHCl. Therefore, DOPA not only adds to CXMS a class of fast, nonhydrolyzable lysine cross-linkers for analysis of protein-protein interactions, but also expands the application of CXMS to analysis of protein folding/unfolding intermediates.