Intrinsic N-Terminal Reactivity and Improved Analysis of DSSO-Carbamate and Carbamate-Based Cross-Linkers

Cross-linking mass spectrometry (XL-MS) has emerged as a powerful approach for probing protein structure and conformational dynamics. Conventional cross-linkers typically contain two <i>N</i>-hydroxysuccinimide (NHS) ester groups that primarily target lysine residues. Here, we report the optimization of the in-solution reactivity of disuccinimidyl sulfoxide carbamate (DSSO-carbamate), an analogue of DSSO in which the two NHS ester groups are replaced by NHS carbamates. The enhanced stability of the carbamate functionality reduces the degradation of DSSO through retro-ene sulfoxide elimination under standard XL-MS buffer conditions, thereby improving cross-linking efficiency. We further characterized the gas-phase dissociation behavior of DSSO-carbamate and optimized the collision energy (CE) parameters for automated data analysis with XL-MS search engines. Mapping of cross-linking sites for bovine serum albumin revealed an unexpectedly high frequency of cross-links involving the protein N-terminus, suggesting increased N-terminal reactivity of NHS carbamates relative to NHS esters. This hypothesis was corroborated by comparative cross-linking of nonacetylated and N-terminally acetylated α-synuclein using DSSO-carbamate and the NHS ester-based disuccinimidyl dibutyric urea (DSBU). We observed the same reactivity trend for the NHS carbamate-based cross-linker NNP9. Proteome-wide XL-MS analysis confirmed a higher propensity of NHS carbamate-based reagents to form cross-links with protein N-termini compared to NHS ester-based cross-linkers. Together, these results show that NHS carbamate-based reagents provide complementary XL-MS restraints to NHS ester-based cross-linkers and are particularly useful for investigating systems where N-terminal interactions are functionally relevant. We anticipate that this unique N-terminal selectivity of NHS carbamates will find broader applications in bioconjugation and chemical proteomics.