Quantitative Mapping of NHS Ester–Protein Reactivity Using Native Top-Down Mass Spectrometry

Journal article

Covalent ligands are widely used to label, probe, and modulate proteins, but peptide-centric readouts obscure how modifications colocalize on intact proteoforms. This can limit insight into ligand mechanism, modification stoichiometry, and the architecture of multisite protein conjugates. We present a general native top-down mass spectrometry workflow that quantifies electrophile reactivity directly on intact proteins. Using NHS esters as a model electrophile class, we apply a deconvolution framework to infer differential reactivity at primary amines across promiscuous, multisite modification patterns. The approach preserves full modification connectivity, avoids sample-preparation artifacts associated with denaturation and digestion, and should extend to electrophiles with unknown reactivity. Overall, this framework provides a general platform for designing covalent therapeutics, bioconjugates, and activity-based probes with proteoform-level resolution.

Authors: Bennett, JL; Ramsay, OB; Lutomski, CA; Kirschbaum, C; Robinson, CV

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: American Chemical Society
• Journal: Journal of The American Society for Mass Spectrometry
• Volume: 37
• Issue: 3
• Pages: 702-709
• Publication date: 2026-01-28
• Acceptance date: 2026-01-23
• DOI: 10.1021/jasms.5c00395
• EISSN: 1879-1123
• ISSN: 1044-0305
• Language: English
• Keywords: NHS esters; covalent modification; intact proteins; native top-down mass spectrometry; electrophile reactivity; proteoform analysis