Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments

Anal Chem. 2019 Sep 3;91(17):10970-10978. doi: 10.1021/acs.analchem.9b00973. Epub 2019 Aug 13.


Insight into the structure-function relationship of membrane proteins is important to understand basic cell function and inform drug development, as these are common targets for drugs. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established technique for the study of protein conformational dynamics and has shown compatibility with membrane proteins. However, the digestion and mass analysis of peptides from membrane proteins can be challenging, severely limiting the HDX-MS experiment. Here we compare the digestion of four integral membrane proteins-Cl-/H+ exchange transporter (ClC-ec1), leucine transporter (LeuT), dopamine transporter (DAT), and serotonin transporter (SERT)-by the use of porcine pepsin and three alternative aspartic proteases either in-solution or immobilized on-column in an optimized HDX-MS-compatible workflow. Pepsin was the most favorable for the digestion of ClC-ec1 and LeuT, providing coverage of 82.2 and 33.2% of the respective protein sequence; however, the alternative proteases surpassed pepsin for the digestion of DAT and SERT. By also screening quench solution additives, we observe that the denaturant urea was beneficial, resulting in improved sequence coverage of all membrane proteins, in contrast to guanidine hydrochloride. Furthermore, significant improvements in sequence coverage were achieved by tailoring the chromatography to handle hydrophobic peptides. Overall, we demonstrate that the susceptibility of membrane proteins to proteolytic digestion during HDX-MS is highly protein-specific. Our results highlight the importance of having multiple proteases and different quench buffer additives in the HDX-MS toolbox and the need to carefully screen a range of digestion conditions to successfully optimize the HDX-MS analysis of integral membrane proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antiporters / analysis*
  • Antiporters / chemistry
  • Aquifex
  • Aspartic Acid Proteases / chemistry
  • Bacteria
  • Dopamine Plasma Membrane Transport Proteins / analysis*
  • Dopamine Plasma Membrane Transport Proteins / chemistry
  • Drosophila Proteins / analysis*
  • Drosophila Proteins / chemistry
  • Drosophila melanogaster
  • Escherichia coli
  • Escherichia coli Proteins / analysis*
  • Escherichia coli Proteins / chemistry
  • Humans
  • Hydrogen Deuterium Exchange-Mass Spectrometry / methods*
  • Models, Molecular
  • Pepsin A / chemistry
  • Peptide Fragments / analysis*
  • Proteolysis
  • Serotonin Plasma Membrane Transport Proteins / analysis*
  • Serotonin Plasma Membrane Transport Proteins / chemistry
  • Structure-Activity Relationship
  • Swine
  • Urea / chemistry


  • Antiporters
  • CLC-ec1 protein, E coli
  • DAT protein, Drosophila
  • Dopamine Plasma Membrane Transport Proteins
  • Drosophila Proteins
  • Escherichia coli Proteins
  • Peptide Fragments
  • SLC6A4 protein, human
  • Serotonin Plasma Membrane Transport Proteins
  • Urea
  • Aspartic Acid Proteases
  • Pepsin A

Supplementary concepts

  • Aquifex aeolicus