# 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.

## Key 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.