2026-10-11T18:15:48Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34018254 2026-09-28T19:05:12Z category_1 category_4 category_915 category_7 category_16 category_873 category_734 category_132 category_135 portal_63 item_type_3 month_year_09_2026

Droplet Microfluidic Hydrogen/Deuterium Exchange for Investigating Protein Dynamics with Millisecond Precision Dietmar Hammerschmid (14515218) Jakub Sys (25133094) Alistair Bailey (524997) Simon I. R. Lane (17701293) Max Saito (25133097) Theo Hornsey (25133100) Niall Hanrahan (11529412) Andy van Hateren (524998) Howard Broughton (1616962) Alfonso Espada (1900177) Eamonn Reading (1405705) Jonathan West (18255) Biophysics Biochemistry Physical Sciences not elsewhere classified Medicine Physiology Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Infectious Diseases Computational Biology stably folded protein investigate droplet microfluidics highly dynamic regions high diffusive flux droplet microfluidic hydrogen rapid droplet merging rapid conformational switching millisecond precision hydrogen investigating protein dynamics fast uptake characteristics droplet hdx processors 2 </ sub 2 +</ sup single millisecond incubations millisecond hdx enabled fast dynamics rapid mixing uniform incubations scale incubations triggered allostery room temperature pd 7 mass spectrometry limiting step heightened plasticity hand ca findings demonstrate deuterium exchange detected within defined ef conformation changes binding motifs acid quenching 10 milliseconds Hydrogen/Deuterium eXchange (HDX) methods for studying protein dynamics would benefit from millisecond-scale incubations to probe intrinsically disordered proteins, highly dynamic regions, and conformation changes. 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. With the ability to access millisecond time scales, the fast dynamics of calmodulin, a model of calcium-triggered allostery with rapid conformational switching, was investigated. At 10 milliseconds, we could observe significant deuterium uptake within the well-defined EF-hand Ca²⁺ binding motifs. These findings demonstrate that millisecond HDX enabled by droplet microfluidics allows areas of heightened plasticity to be detected within a stably folded protein. 2026-09-28T00:00:00Z Dataset Dataset 10.1021/acs.analchem.6c02766.s003 https://figshare.com/articles/dataset/Droplet_Microfluidic_Hydrogen_Deuterium_Exchange_for_Investigating_Protein_Dynamics_with_Millisecond_Precision/34018254 CC BY-NC 4.0