NHS Carbamate Cross-Linkers Provide Complementary XL-MS Restraints via Preferential N-Terminal Targeting

ABSTRACT: Cross-linking mass spectrometry (XL-MS) has become a valuable tool for investigating the structural morphology and plasticity of proteins. Traditional cross-linkers contain two N-hydroxy succinimide (NHS) esters that mainly react with lysine residues. In this work, we optimized the in-solution reactivity of the cross-linker disuccinimidyl sulfoxide (DSSO) carbamate. DSSO carbamate is a DSSO analogue bearing two NHS carbamate groups instead of NHS esters as reactive moiety. The higher stability of the carbamate function mitigates the degradation of DSSO via retro-ene sulfoxide elimination in standard XL-MS buffers which limits the cross-linking yield. Additionally, we elucidated its characteristic gas-phase dissociation behavior and optimized the collision energy (CE) for automated analysis with XL-MS search engines. Cross-link site analysis of bovine serum albumin highlighted an unexpected abundance of cross-link species involving the N-terminus of the protein. We attributed this to a higher N-terminal reactivity of the NHS carbamate group compared to NHS esters. We confirmed our hypothesis by cross-linking non-acetylated and N-terminally acetylated α-asynuclein with DSSO carbamate and the NHS ester-based disuccinimidyl dibutyric urea (DSBU) cross-linker. Finally, we applied the NHS carbamate-based cross-linker NNP9 and observed the same chemical propensity. NHS carbamate-based reagents give complementary XL-MS restraints to NHS ester-based cross-linkers and can be useful for studying systems where N-terminal binding plays a significant role. We anticipate that this unexpected selectivity of NHS carbamates to protein N-termini may have applications in bioconjugation and chemical proteomics in general.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Alessio Di Ianni  

LAB HEAD: Andrea Sinz

PROVIDER: PXD069252 | Pride | 2026-03-30

REPOSITORIES: Pride