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 N-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.