Hydrogen Deuterium Exchange Coupled with SELECT SERIES Cyclic IMS Mass Spectrometer for Higher Workflow Efficiency

Importance of Hydrogen-Deuterium Exchange Mass Spectrometry

Hydrogen-deuterium exchange mass spectrometry is a key technique for probing protein higher order structure, dynamics, and binding interactions. It complements high resolution methods such as NMR, cryo-EM, and X-ray crystallography by offering faster throughput and cost efficiency, making it valuable for epitope mapping, drug candidate screening, and development of therapeutic biomolecules.

Objectives and Study Overview

This study evaluates the performance gains of the new SELECT SERIES Cyclic IMS mass spectrometer for HDX workflows. Benchmarking against SYNAPT G2-Si and SYNAPT XS instruments, it investigates peptide identification rates, sequence coverage, dynamic range, and the feasibility of lower sample loads and shorter chromatographic gradients.

Methodology and Instrumentation

Phosphorylase B standards at 8, 16, and 32 µM were labeled by dilution into deuterated buffer, quenched, digested on a pepsin column, and separated by low-temperature LC. Peptides were analyzed in data-independent HDMSE mode with one or two passes of the ion mobility cell. Key LC conditions included a 1 x 100 mm BEH C18 column at 0 °C and a 7-minute gradient at 40 µL/min.

Used Instrumentation

• SELECT SERIES Cyclic IMS mass spectrometer with dual gain ADC and scalable ion mobility resolution
• ACQUITY UPLC M-Class system with HDX automation
• Enzymate BEH pepsin column and VanGuard BEH C18 trap
• MassLynx, Quartz, PLGS, and DynamX software for data acquisition and analysis

Main Results and Discussion

The Cyclic IMS delivered 560 identified peptides and 100% sequence coverage for a 32 µM sample, outperforming SYNAPT XS (360 peptides, 98.5% coverage) and G2-Si (240 peptides, 89% coverage). The dual gain ADC extended dynamic range and minimized saturation for abundant ions, improving isotope accuracy. At reduced loads (10–40 pmol injections), the system maintained over 90% coverage, demonstrating robust performance for sample-limited applications.

Benefits and Practical Applications

• 30% more peptide identifications than previous generation
• Accurate isotope measurement under high ion currents
• Ability to reduce sample load fourfold while preserving coverage
• Potential to shorten gradients for higher daily throughput and lower cost

Future Trends and Potential Applications

Advances in cyclic ion mobility and dual gain detection are expected to further improve HDX workflows. Future work may explore even shorter gradients, integration with automated sample handling, and applications to larger protein complexes and membrane proteins. Enhanced informatics will streamline data processing for highly complex spectra.

Conclusion

The SELECT SERIES Cyclic IMS significantly enhances HDX-MS performance, providing higher peptide coverage, improved sensitivity, and dynamic range. These advances support more efficient, cost-effective protein structural analysis, with promising applications in biopharmaceutical research and quality control.

References

1. Puchades C et al Epitope Mapping of Diverse Influenza Hemagglutinin Drug Candidates using HDX-MS Sci Rep 9 4735 2019
2. 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 720006870EN
3. Sørensen L Salbo R Optimized Workflow for Selecting Peptides for HDX-MS Data Analyses J Am Soc Mass Spectrom 29 2278–2281 2018