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Peer-reviewedCC BYAnalytical chemistry

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

  • Dietmar Hammerschmid1
  • Jakub Sys1
  • Alistair Bailey2,3
  • Simon I R Lane4,3
  • Max Saito2
  • Theo Hornsey1
  • Niall Hanrahan4,3
  • Andy van Hateren2,3
  • Howard Broughton5
  • Alfonso Espada5
  • Eamonn Reading1,3
  • Jonathan West2,3
5 affiliations
  1. 1School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, SouthamptonSO17 1BJ, U.K.
  2. 2Cancer Sciences, Faculty of Medicine, University of Southampton, SouthamptonSO17 1BJ, U.K.
  3. 3Institute for Life Sciences, University of Southampton, SouthamptonSO17 1BJ, U.K.
  4. 4School of Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, SouthamptonSO17 1BJ, U.K.
  5. 5Centro de Investigación Lilly, SA, Avenida de la Industria 30, Alcobendas28108, Spain.
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Abstract

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.

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Cite

BibTeXRIS
BibTeX
@article{hammerschmid2026droplet,
  title = {{Droplet Microfluidic Hydrogen/Deuterium Exchange for Investigating Protein Dynamics with Millisecond Precision.}},
  author = {Dietmar Hammerschmid and Jakub Sys and Alistair Bailey and Simon I R Lane and Max Saito and Theo Hornsey and Niall Hanrahan and Andy van Hateren and Howard Broughton and Alfonso Espada and Eamonn Reading and Jonathan West},
  journal = {Analytical chemistry},
  year = {2026},
  month = oct,
  doi = {10.1021/acs.analchem.6c02766},
  url = {https://europepmc.org/article/MED/42916134},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Droplet Microfluidic Hydrogen/Deuterium Exchange for Investigating Protein Dynamics with Millisecond Precision.
AU  - Dietmar Hammerschmid
AU  - Jakub Sys
AU  - Alistair Bailey
AU  - Simon I R Lane
AU  - Max Saito
AU  - Theo Hornsey
AU  - Niall Hanrahan
AU  - Andy van Hateren
AU  - Howard Broughton
AU  - Alfonso Espada
AU  - Eamonn Reading
AU  - Jonathan West
PY  - 2026
DA  - 2026/10/01
JO  - Analytical chemistry
DO  - 10.1021/acs.analchem.6c02766
UR  - https://europepmc.org/article/MED/42916134
AB  - 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.
SN  - 0003-2700
SN  - 1520-6882
ER  -