Abstract
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.