# Hydrogen‐Deuterium Exchange Mass Spectrometry for Molecular Glue Characterization

## TL;DR

This paper introduces hydrogen-deuterium exchange mass spectrometry as a tool to study how molecular glues stabilize protein interactions.

## Contribution

The paper demonstrates the use of HDX-MS to reveal binding sites and conformational changes in molecular glue-induced protein complexes.

## Key findings

- HDX-MS identifies molecular glue binding sites and conformational changes in protein complexes.
- The method shows how molecular glues differentially stabilize protein interactions.
- The approach is applicable to the 14-3-3 protein system with fusicoccin A as a molecular glue.

## Abstract

Molecular glues are powerful bioactive molecules that stabilize protein–protein interactions. Yet, the precise mechanisms by which many molecular glues exert their adhesive effects are still not well understood. Native mass spectrometry is an established technique used to monitor the stoichiometry and binding equilibria of molecular glue‐induced protein–protein interactions. However, knowledge is lacking on how protein interaction dynamics change upon molecular glue‐induced stabilization, and what conformational changes occur that enhance the stability of the resulting protein‐protein‐glue ternary complex. Here, hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) is showcased as an analytical tool for molecular glue characterization. Using a broadly applicable molecular glue system involving the eukaryotic regulatory protein 14‐3‐3, its binding partners, and the molecular glue fusicoccin A, the power of HDX‐MS is shown in revealing not only molecular glue binding sites, but also conformational changes upon glue binding that result in differentially stabilized protein‐protein complexes. Overall, the HDX‐MS approach will become highly envisaged and valuable for the characterization of molecular glues, guiding their optimization toward successful design.

Molecular glues that stabilize protein–protein interactions are challenging to identify. Here, hydrogen‐deuterium exchange mass spectrometry can show and reveal gluing mechanisms that vary between protein binding partners.