Apparatus for Automated Continuous Hydrogen Deuterium Exchange Mass Spectrometry Measurements from Milliseconds to Hours

[Image: see text] Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a rapidly growing technique for protein characterization in industry and academia, complementing the “static” picture provided by classical structural biology with information about the dynamic structural changes that accompany biological function. Conventional hydrogen deuterium exchange experiments, carried out on commercially available systems, typically collect 4–5 exchange timepoints on a timescale ranging from tens of seconds to hours using a workflow that can require 24 h or more of continuous data collection for triplicate measurements. A small number of groups have developed setups for millisecond timescale HDX, allowing for the characterization of dynamic shifts in weakly structured or disordered regions of proteins. This capability is particularly important given the central role that weakly ordered protein regions often play in protein function and pathogenesis. In this work, we introduce a new continuous flow injection setup for time-resolved HDX-MS (CFI-TRESI-HDX) that allows automated, continuous or discrete labeling time measurements from milliseconds to hours. The device is composed almost entirely of “off-the-shelf” LC components and can acquire an essentially unlimited number of timepoints with substantially reduced runtimes compared to conventional systems.