Droplet Microfluidic Hydrogen/Deuterium Exchange for Investigating Protein Dynamics with Millisecond Precision

Here, we investigate droplet microfluidics for rapid mixing to trigger D₂O labeling, uniform incubations, and rapid droplet merging for acid quenching in advance of mass spectrometry. A surfactant-free merging approach combining expansion elements for synchronized droplet collision proved robust.

The high diffusive flux of D₂O and protons enables microsecond mixing to trigger and arrest D₂O labeling, respectively, affording the possibility of single millisecond incubations. Droplet HDX processors were used to measure the fast uptake characteristics of a model peptide. Forward exchange measurements demonstrate D₂O labeling to be the rate-limiting step, in essence defining 10 milliseconds as the minimum practical incubation time for proteins at room temperature, pD 7.4.