PRESSURIZED ONLINE PEPSIN DIGESTION OF PROTEINS FOR HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY

Importance of Topic

Hydrogen/deuterium exchange mass spectrometry (HDX MS) is a key technique for probing protein structure and dynamics. Efficient and reproducible proteolytic digestion, typically achieved by online pepsin digestion, is essential to maximize sequence coverage and spatial resolution. However, conventional low-pressure pepsin digestion often yields insufficient peptide generation for challenging targets such as monoclonal antibodies. This study presents a pressurized online pepsin digestion approach to enhance peptide yield and resolution while maintaining minimal back-exchange.

Objectives and Study Overview

The main goals of this work were to compare digestion efficiency of a monoclonal IgG2 under normal (≈1,000 psi) and elevated pressures (≈12,000–15,000 psi), optimize digestion parameters (temperature, flow rate, quench hold time, chaotropic concentration), and assess deuterium back-exchange under each condition. The study aimed to demonstrate improved sequence coverage, shorter peptide fragments, and compatible reagent usage for downstream HDX MS analysis.

Methodology

Samples included denosumab IgG2 diluted into deuterated buffer and fully deuterated standard peptides (angiotensin II, bradykinin). After labeling, quenching was performed with chilled buffer containing guanidine HCl and TCEP. Key variables evaluated:

• Digestion pressure: normal vs high (~12,000–15,000 psi)
• Digestion temperature: room temperature to 15 °C
• Quench hold time: 1–5 min at 0 °C
• Flow rates and desalting time: 100–200 µL/min, 1–4 min
• Chaotropic concentrations: 3–4 M GdnHCl, 0.4–0.5 M TCEP

Used Instrumentation

The workflow employed:

• Waters M-Class UPLC system with HDX Manager at 0 °C
• Prototype back pressure regulator to generate up to 15,000 psi
• Sustainable BEH column immobilized with pepsin
• Waters Synapt G2-Si HDMS for peptide detection
• PLGS 3.0.2 and DynamX 3.0 software for peptide identification and mapping

Main Results and Discussion

High-pressure digestion delivered significantly higher peptide redundancy and sequence coverage for both heavy and light chains of IgG2 compared to normal pressure. Key findings:

• More overlapping and shorter peptides under high pressure improved spatial resolution.
• Optimal digestion at 15 °C with a 5 min quench hold minimized back exchange while maximizing coverage.
• High flow rate (200 µL/min) and extended desalting reduced salt contamination without increasing deuterium loss.
• Comparable or slightly improved digestion efficiency achieved using lower chaotrope (3 M GdnHCl, 0.4 M TCEP) under high pressure versus higher concentrations under normal pressure.
• Deuterium loss for standard peptides remained low and only marginally higher under high pressure, confirming suitability for HDX MS.

Benefits and Practical Applications

This pressurized online pepsin digestion method offers:

• Enhanced reproducibility and peptide generation for challenging proteins such as mAbs
• Greater sequence coverage and improved resolution of exchange sites
• Reduced use of chaotropic agents, facilitating downstream handling
• Compatibility with automated HDX workflows for structural and biopharmaceutical analyses

Future Trends and Potential Applications

Building on these results, future directions include:

• Extension to other proteases and diverse protein classes under high pressure
• Integration with advanced automation and microfluidics for HDX MS
• Development of novel immobilized enzyme columns optimized for pressure-enhanced digestion
• Application to mapping protein–ligand interactions and conformational changes in biotherapeutics

Conclusion

Pressurized online pepsin digestion represents a robust enhancement to HDX MS workflows, delivering improved sequence coverage, finer spatial resolution, and efficient reagent usage. This strategy is particularly valuable for the structural analysis of large and disulfide-rich proteins such as monoclonal antibodies.

References

• Ahn J. et al. Anal. Chem. 2012, 84, 7256–7262.
• Wu Y. et al. Anal. Chem. 2006, 78, 1719–1723.