Probe the Protein Conformation using Top-down Hydrogen Exchange Mass Spectrometry at Higher Resolution with Electron Transfer Dissociation

Importance of the Topic

The combination of hydrogen/deuterium exchange mass spectrometry (HDX-MS) with electron transfer dissociation (ETD) offers a powerful approach to probe protein structure at single residue resolution. This capability is critical for understanding conformational dynamics, ligand binding, and allosteric regulation in proteins of interest across biotechnology, drug discovery, and fundamental biochemical research.

Objectives and Study Overview

This study aimed to optimize HDX-ETD workflows for minimal deuterium scrambling, validate the protocol with a model peptide, and apply both top-down and bottom-up HDX-ETD analyses to characterize conformational changes in the calcium-binding protein calmodulin in its apo- and holo- forms.

Methodology

• Calibration using a fully deuterated synthetic peptide (P1: HHHHHHIIKIIK) to evaluate ETD parameters and quantify scrambling.
• Bottom-up HDX-ETD: labeling, proteolytic digestion, followed by LC-ETD MS/MS to achieve high sequence coverage.
• Top-down HDX-ETD: direct infusion of denatured intact calmodulin into the Orbitrap Ascend instrument to capture fragment ions across the sequence.
• Time-course deuterium labeling at multiple time points to compare uptake kinetics in apo- and holo- states.

Used Instrumentation

• Thermo Scientific Orbitrap Ascend Structural Biology Edition mass spectrometer equipped with ETD capability
• C18 trap column and Hypersil GOLD C18 analytical column for desalting and separation
• Automated HDX workstation for precise temperature and labeling control
• BioPharma Finder 5.2 and HDExaminer 3.4.1 software for peptide identification and deuterium uptake analysis

Main Results and Discussion

• Optimized ETD settings achieved negligible deuterium scrambling in the model peptide, enabling near single-residue resolution.
• Bottom-up experiments provided ~100% sequence coverage for both apo- and holo- calmodulin, identifying 140 peptides.
• Top-down ETD yielded ~88% coverage and revealed consistent uptake trends with the bottom-up approach.
• Apo- calmodulin exhibited higher deuterium incorporation overall, with pronounced differences in specific binding regions (residues 21-32, 57-68, 94-105, 130-141).
• Detailed fragment ion uptake curves demonstrated the ability to localize conformational changes at the single-amino-acid level.

Benefits and Practical Applications

• Provides a robust protocol for achieving minimal hydrogen scrambling in HDX-ETD experiments.
• Enables high-resolution mapping of protein dynamics and ligand-induced conformational shifts.
• Applicable to biopharmaceutical characterization, quality control of protein therapeutics, and mechanistic studies of enzyme regulation.

Future Trends and Applications

• Integration of ion mobility separation to further reduce spectral complexity and enhance fragment ion resolution.
• Expansion to larger protein assemblies and membrane proteins through advanced sample handling and gas-phase separation.
• Development of AI-driven data analysis pipelines to streamline deuterium uptake quantitation and structural interpretation.
• Combination with complementary structural methods (cryo-EM, NMR) for multi-scale characterization of dynamic systems.

Conclusion

The optimized HDX-ETD workflow described here delivers high sequence coverage and near single-residue resolution with minimal deuterium scrambling. Application to calmodulin revealed distinct conformational differences between apo- and holo- forms, demonstrating the method's power for detailed protein structural analysis.

References

1. Kasper D. Rand and Thomas J. D. Jørgensen. Analytical Chemistry 2007, 79(22), 8686–8693. DOI: 10.1021/ac0710782
2. Kasper D. Rand, Martin Zehl, Ole N. Jensen, and Thomas J. D. Jørgensen. Analytical Chemistry 2010, 82(23), 9755–9762. DOI: 10.1021/ac101889b