Probe the protein conformation using top-down hydrogen exchange mass spectrometry at higher resolution with electron transfer dissociation

Importance of the topic

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a vital technique for probing protein conformations and dynamics under near-native conditions. Achieving single-residue resolution through top-down fragmentation enhances our understanding of protein folding, ligand binding, and structural changes that occur upon complex formation.

Objectives and study overview

This work aims to optimize electron transfer dissociation (ETD) conditions on an Orbitrap Ascend Structural Biology Edition mass spectrometer to minimize hydrogen scrambling and back-exchange, enabling high-resolution mapping of deuterium incorporation. Using a model peptide (P1) for method development, the study then applies both top-down and bottom-up HDX-ETD workflows to compare conformational differences between apo- and holo-forms of calmodulin.

Methodology and instrumentation

The HDX workflow employed the Thermo Scientific Vanquish Flex LC system coupled to the Orbitrap Ascend instrument. For scrambling optimization, fully deuterated P1 peptide (HHHHHHIIKIIK) was analyzed under varied source temperatures and ion transfer voltages to identify settings that preserve deuterium labeling. Bottom-up experiments combined simultaneous full-scan and ETD MS2 during LC separation of quenched, pepsin-digested calmodulin peptides. Top-down analyses directly infused denatured protein through a C18 trap to trigger ETD fragmentation of intact calmodulin.

Main results and discussion

• Optimal ETD parameters on the Orbitrap Ascend yielded negligible hydrogen scrambling for P1, confirmed by consistent deuterium levels in c- and z-ions and ammonia loss reporters.
• Bottom-up ETD achieved nearly 100 % sequence coverage for both apo- and holo-calmodulin, while top-down ETD covered 88 % of the sequence.
• Apo-calmodulin exhibited higher global deuterium uptake than the Ca2+-bound form, reflecting increased solvent accessibility in the absence of calcium.
• Region-specific HDX profiles revealed four EF-hand Ca2+ binding sites (residues 21–32, 57–68, 94–105, 130–141) with reduced exchange upon calcium binding.
• Combining top-down and bottom-up data enabled near single-residue resolution for N-terminal and selected C-terminal segments, illustrating the power of ETD to localize deuterium incorporation.
• Top-down ETD differentiated calmodulin variants: incorporation of an N-terminal His-tag increased charge distribution and improved coverage of N-terminal fragments at the cost of C-terminal coverage.

Benefits and practical applications

HDX-ETD on the Orbitrap Ascend platform provides a robust approach for structural characterization of proteins and complexes with higher spatial resolution than conventional HDX-MS. The combined top-down/bottom-up strategy delivers comprehensive conformational maps, supporting studies in biopharmaceutical quality control, protein engineering, and drug discovery.

Future trends and applications

Advances may include integration of automated high-throughput HDX-ETD workflows, expansion to larger protein assemblies, and incorporation of alternative non-ergodic fragmentation methods. Coupling HDX-ETD with ion mobility or ultraviolet photodissociation could further enhance resolution and throughput for ligand-binding and allosteric mechanism studies.

Conclusion

Optimized ETD parameters on the Orbitrap Ascend instrument enable minimal scrambling and near single-residue HDX analysis. Both top-down and bottom-up ETD workflows reliably distinguish conformational differences between apo- and holo-calmodulin, validating the approach for detailed protein structural studies.

References

1. Rand KD, Jørgensen TJD Analytical Chemistry 2007 79(22):8686–8693 DOI:10.1021/ac0710782
2. Rand KD, Zehl M, Jensen ON, Jørgensen TJD Analytical Chemistry 2010 82(23):9755–9762 DOI:10.1021/ac101889b