Enhanced Performance of the SYNAPT XS and Its Impact on Hydrogen Deuterium Exchange Mass Spectrometry (HDX MS) Data Quality

Importance of the topic

Hydrogen–deuterium exchange mass spectrometry (HDX MS) provides a powerful approach to interrogate protein structure and dynamics at the peptide level. This technique is widely applied in biopharmaceutical development, for epitope mapping, stability studies, and investigating disease-related conformational changes.

Goals and Overview of the Study

This technology brief evaluates how the enhanced StepWave XS ion guide and extended flight tube of the SYNAPT XS high-resolution mass spectrometer improve sensitivity, mass resolution, and hydrogen/deuterium back-exchange performance in HDX MS experiments.

Methodology and Instrumentation

HDX workflow included rapid online proteolysis using an Enzymate BEH pepsin column, high-pressure UPLC separation, and data-independent acquisition with ion mobility separation. The SYNAPT XS instrument features a third-generation StepWave XS ion guide to optimize ion transfer and reduce energy deposition, plus a 45 cm longer time-of-flight path for enhanced mass resolution.

Main Results and Discussion

• Peptide identifications and sequence coverage increased on SYNAPT XS compared to SYNAPT G2-Si in both sensitivity and resolution modes.
• Extended flight path and ion mobility improved peak capacity, enabling clearer separation of isotopic clusters.
• Back-exchange experiments with yeast enolase demonstrated reduced in-source H/D back-exchange, reflected by higher measured deuteration across susceptible peptides.

Benefits and Practical Applications

The improved sensitivity and resolution support more confident peptide assignment and deeper sequence coverage, critical for structural proteomics, biopharmaceutical comparability, and analysis of complex protein systems.

Future Trends and Potential Applications

Ongoing developments may include further automation, integration with high-throughput platforms, and advanced data analysis tools for large-scale proteome dynamics studies. The enhanced performance of instruments like SYNAPT XS will facilitate exploration of larger, more complex assemblies and real-time structural changes.

Conclusion

The SYNAPT XS mass spectrometer, with its StepWave XS ion guide and extended flight tube, delivers significant gains in sensitivity, resolution, and reduced back-exchange for HDX MS. These improvements translate to greater confidence in structural measurements and broaden the scope of protein dynamics research.

Reference

• Sorensen L. et al. Optimized Workflow for Selecting Peptides for HDX-MS Data Analyses. J. Am. Soc. Mass Spectrom. 2018, 29, 2278–2281.