Multiple ion activations and proton transfer charge reduction facilitate the characterization of capsid proteoforms of recombinant adeno-associated virus 6 (rAAV6)

Significance of the Topic

The detailed characterization of adeno-associated virus (AAV) capsid proteins is critical for the development and quality assurance of gene therapy vectors. Accurate profiling of the VP1, VP2, and VP3 proteoforms informs on capsid assembly, potency, and potential immunogenicity. Advances in mass spectrometry techniques enable in-depth proteoform analysis, supporting regulatory compliance and therapeutic efficacy.

Aims and Study Overview

This study aimed to enhance sequence coverage of the three AAV6 capsid proteins (VP1, VP2, VP3) by integrating multiple gas-phase ion activation methods with proton transfer charge reduction (PTCR). The work focused on comparing higher-energy collisional dissociation (HCD), electron transfer dissociation (ETD), and extended ETD (EThcD) at the MS2 level, combined with PTCR at the MS3 level, to resolve complex fragmentation spectra and extend proteoform mapping.

Methodology and Instrumentation

Purified AAV6 particles were disassembled into individual VP subunits under denaturing conditions. Chromatographic separation utilized reversed-phase UPLC with a C4 column and a linear gradient of aqueous and organic phases containing difluoroacetic acid. Mass spectrometric analysis was conducted on an Orbitrap Eclipse Tribrid platform, performing targeted MS2 experiments with HCD, ETD, and EThcD, followed by PTCR MS3 using perfluoroperhydrophenanthrene anions. Data interpretation employed isotope-fitting algorithms to match fragment clusters and manual validation for sequence assignments.

Main Results and Discussion

Sequence coverage varied across subunits: HCD and ETD MS2 provided limited coverage for VP1 (20%) and VP2 (16%), whereas VP3 achieved higher coverage due to greater abundance. Implementing PTCR MS3 reduced spectral congestion, allowing detection of larger fragments and boosting VP3 coverage to 37.9%. Spectral averaging over multiple charge states improved signal-to-noise ratios. However, PTCR was less effective for the lower abundance VP1 and VP2 due to reduced intensity and overlapping elution profiles.

Benefits and Practical Applications

• Enhanced proteoform mapping improves batch-to-batch consistency monitoring.
• Integration of complementary fragmentation techniques yields more complete capsid protein profiles.
• PTCR reduces spectral overlap, facilitating identification of high-mass fragments.

Future Trends and Opportunities

Emerging separation strategies, such as hydrophobic interaction chromatography, may improve VP1/VP2 resolution, enhancing PTCR applicability. Further optimization of ion-ion reaction conditions and development of automated data analysis pipelines could accelerate high-throughput AAV proteoform characterization. Integration with native MS approaches promises deeper insights into intact capsid heterogeneity.

Conclusion

The combination of HCD, ETD/EThcD MS2 with PTCR MS3 on an Orbitrap Tribrid platform significantly improves the sequence coverage of AAV6 capsid proteins, particularly for the abundant VP3 subunit. While challenges remain for low-abundance proteoforms, this multi-technique workflow sets a foundation for robust proteoform analysis in gene therapy vector development.

Instrumentation Used

• Ultimate 3000 UPLC system with Protein BEH C4 column
• Orbitrap Eclipse Tribrid mass spectrometer with HCD, ETD, EThcD, and PTCR capabilities

References

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