HIGHER SENSITIVITY, DATA QUALITY AND THROUGHPUT FOR HYDROGEN DEUTERIUM EXCHANGE EXPERIMENTS WITH EXTENDED ION MOBILITY SEPARATION

Significance of the Topic

Hydrogen–deuterium exchange mass spectrometry (HDX MS) is a cornerstone technique for probing protein higher order structure, dynamics and interaction sites. Enhanced sensitivity, resolution and throughput in HDX MS are essential for characterizing large protein complexes, supporting therapeutic development, and enabling high-throughput epitope screening.

Objectives and Study Overview

This study evaluates the performance of the new SELECT Series Cyclic IMS system against the Synapt G2-Si and Synapt XS platforms in online HDX MS experiments. Key aims include assessment of peptide identification rates, sequence coverage, spectral quality under truncated chromatographic gradients and reduced sample loads.

Methodology and Used Instrumentation

Automated HDX workflows were performed on a LEAP HDX-2 robot coupled to an ACQUITY M-Class HDX system with an online pepsin column. Peptides were analyzed by HDMSE (data-independent acquisition) on a SELECT Series Cyclic IMS mass spectrometer with variable ion mobility resolution (one-pass or two-pass separation), dual-gain ADC detection and identical ion source settings used for comparison with Synapt G2-Si and XS systems.

Main Results and Discussion

• Cyclic IMS delivered approximately 560 filtered peptides from phosphoylase B, compared to 240 on the G2-Si and 360 on the XS, corresponding to superior sequence coverage (>98%).
• Dual-gain ADC detection improved dynamic range, reducing isotope saturation at high ion currents and enhancing accuracy of deuterium uptake measurements.
• Truncated LC gradients (3 min analytical run) maintained >90 % sequence coverage even at fourfold reduced load (10 fmol), demonstrating robust throughput gains.
• Variable ion mobility separation resolved overlapping isotope patterns arising from coeluting peptides, particularly under shortened gradients.

Benefits and Practical Applications

• Increased peptide identification and coverage enable more detailed mapping of protein dynamics and interaction interfaces.
• Enhanced sensitivity reduces sample requirements and lowers per-injection costs.
• Truncated gradients enable up to 2–3× higher throughput without compromising data quality, supporting large-scale epitope screening or QA/QC workflows.

Future Trends and Applications

Advances in cyclic ion mobility resolution are expected to further improve separation of complex peptide mixtures, enabling HDX studies on larger assemblies. Integration with machine-learning algorithms for spectral deconvolution and automated data analysis will streamline high-throughput structural proteomics and drug discovery campaigns.

Conclusion

The SELECT Series Cyclic IMS system substantially outperforms previous-generation instruments for HDX MS by delivering greater sensitivity, improved spectral quality and higher throughput. These advances allow reliable protein dynamic analysis with reduced sample loads and shortened chromatographic runs, offering significant benefits for structural biology and biopharmaceutical research.

Reference

• Morrison L, Anderson M, Quinn C. Enhanced Performance of the SYNAPT XS and Its Impact on Hydrogen Deuterium Exchange Mass Spectrometry (HDX MS) Data Quality. Waters Technical Note 720006870, 2020.
• Sørensen L, Salbo R. Optimized Workflow for Selecting Peptides for HDX-MS Data Analyses. J Am Soc Mass Spectrom. 2018;29:2278–2281.