HydroBot: Software for Interactive Hydrogen/Deuterium-Exchange Mass Spectrometry Multistate Analysis

Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful technique for probing protein dynamics, stability, and interactions. However, multistate and nonequilibrium experiments currently do not have available analysis tools. We present HydroBot, a software designed for comprehensive and interactive HDX-MS data analysis and visualization. HydroBot supports rapid and automated uptake plotting, statistical testing of differences between protein states, and multiple interactive visualization modes including bar plots, Woods plots, and heatmaps. Statistical tools such as volcano plots and error distribution analyses are integrated to assess data robustness, enabling interactive exploration of labeling differences. Correlated structural dynamics are revealed by <i>k</i>-means or hierarchical clustering, facilitating pattern discovery within multistate HDX data for proteins at equilibrium and nonequilibrium. Additionally, scripts are created to enable visualization of HydroBot analyses on protein structures. This user-friendly tool streamlines HDX-MS validation and interpretation from processed data to biologically relevant insights.