Pressurized Online Pepsin Digestion of mAb IgG2 for Hydrogen Deuterium Exchange Mass Spectrometry

Importance of the Topic

Pepsin digestion is critical for hydrogen deuterium exchange mass spectrometry (HDX-MS) as it generates localized peptide fragments that reveal protein conformational dynamics. Efficient online digestion minimizes pepsin autolysis contamination and ensures reproducible results. High-pressure digestion further enhances cleavage of structurally stable proteins, such as monoclonal antibodies, under HDX-compatible conditions.

Study Goals and Overview

This study evaluated pressurized online pepsin digestion of IgG2 on a Waters ACQUITY UPLC M-Class platform with HDX Technology. It aimed to optimize digestion temperature, quench holding time, flow rates, and chaotrope concentration under high (~15,000 psi) versus normal (~1,000 psi) pressure. Key performance metrics included sequence coverage, peptide redundancy, spatial resolution, and deuterium back exchange.

Methodology and Instrumentation

• UPLC System: ACQUITY UPLC M-Class with HDX Technology
• Digestion Column: Enzymate Pepsin Column, BEH immobilized pepsin, 2.1×30 mm, 5 µm
• Trap Column: ACQUITY UPLC BEH C18 VanGuard Pre-Column, 2.1×5 mm, 1.7 µm
• Analytical Column: ACQUITY UPLC BEH C18, 1×100 mm, 1.7 µm
• Mass Spectrometry: SYNAPT G2-Si HDMSE (ESI+, m/z 50–2000)
• Software: ProteinLynx Global SERVER 3.0.2 and DynamX 3.0

Key Results and Discussion

• Digestion Temperature: Raising the column compartment from 0 °C to 15 °C improved sequence coverage and generated more overlapping peptides for both heavy and light chains; 15 °C was selected.
• Quench Holding Time: Extending quench time to 5 min at 0 °C increased digestion efficiency under high pressure compared to normal pressure.
• Flow Rates: An initial loading at 100 µL/min for 1 min followed by desalting at 200 µL/min for 3 min provided cleaner MS backgrounds and adequate digestion time.
• Chaotrope Concentrations: High-pressure digestion enabled reduction of GdnHCl from 4 to 3 M and TCEP from 0.5 to 0.4 M while maintaining or improving efficiency.
• Sequence Coverage: High-pressure digestion increased IgG2 heavy chain coverage from 94% to 97%, with additional unique shorter peptides improving spatial resolution.
• Deuterium Back Exchange: Fully deuterated angiotensin II and bradykinin showed comparable back exchange (~13–15%) under both pressure conditions; digestion temperature and quench time had minor effects.

Benefits and Practical Applications

• Enhanced sequence redundancy and coverage accelerate mapping of antibody higher-order structures.
• Shorter overlapping peptides improve spatial resolution in HDX-MS studies.
• Reduced chaotrope loads simplify sample cleanup and lower MS background noise.
• High-pressure digestion offers a robust solution for proteolysis of disulfide-rich or hydrophobic proteins.

Future Trends and Applications

• Integration of high-pressure digestion into automated HDX workflows for high-throughput structural analysis of antibodies.
• Adapting the approach to alternative proteases and challenging targets, such as membrane proteins.
• Development of sustainable high-pressure enzyme columns for broad proteomic applications.
• Combining with advanced MS acquisition strategies to further minimize deuterium loss and enhance detection sensitivity.

Conclusion

High-pressure online pepsin digestion on an immobilized BEH column significantly improves HDX-MS analysis of monoclonal antibodies by increasing sequence coverage, peptide redundancy, and spatial resolution while maintaining acceptable deuterium retention. Lower reagent concentrations and streamlined workflows enhance compatibility with downstream MS detection, establishing this method as a powerful tool for protein structural characterization.

References

1. Sliva JL. Pressure stability of proteins. Annual Review of Physical Chemistry. 1993;44:89–113.
2. Ahn J, Jung MC, Wyndham K, Yu YQ, Engen JR. Pepsin immobilized on high strength silica particles for continuous flow online digestion at 10,000 psi. Analytical Chemistry. 2012;84:7256–7262.