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

TL;DR

This paper introduces a new mass spectrometry method to map how covalent ligands react with intact proteins, offering detailed insights into modification patterns.

Contribution

A novel native top-down mass spectrometry workflow is introduced to quantify electrophile reactivity on intact proteins.

Findings

The workflow preserves modification connectivity and avoids artifacts from denaturation and digestion.

The method infers differential reactivity at primary amines across multisite modification patterns.

The approach can be extended to electrophiles with unknown reactivity.

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.

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