Characterization of adeno-associated viral proteins proteoforms using a top-down MS approach on a UHPLC-Orbitrap Ascend Tribrid mass spectrometer

Importance of the topic

The detailed characterization of adeno-associated virus (AAV) capsid proteins is critical for ensuring the safety, quality, and efficacy of gene therapy vectors. Site-specific post-translational modifications (PTMs) and truncated proteoforms can influence viral assembly, cellular uptake, and immunogenicity. Advanced top-down mass spectrometry (MS) techniques enable direct analysis of intact viral proteins, facilitating confident localization of PTMs and verification of proteoform sequences.

Goals and overview of the study

This application note demonstrates a targeted top-down LC-MS approach on a Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer to:

• Localize phosphorylation sites on VP2 capsid protein of AAV6.
• Confirm the sequence of a truncated VP3 proteoform (“VP3 clip”).
• Improve sequence coverage by combining multiple dissociation methods: EThcD, HCD, and UVPD.

Methodology and instrument

Sample preparation:

• AAV6 capsids expressed in HEK293 cells were buffer-exchanged into 80:20 H₂O/ACN with 5 mM TCEP and 0.1% formic acid.
• Protein concentration adjusted to ~0.2 µg/µL.

Chromatography:

• UHPLC using a C4 column (1.0 × 100 mm, 300 Å, 1.7 µm) at 80 °C, flow rate 50 µL/min.
• Mobile phases: 0.1% DFA in water (A) and in acetonitrile (B); linear gradient from 20% to 80% B.

Instrument settings:

• Orbitrap Ascend Tribrid with HMRn+, ETD, and UVPD options.
• Intact mass resolution 15,000; top-down MS2 resolution 120,000.
• Targeted precursor m/z 1,441.5 (46+ VP2, co-isolated 30+ VP3 clip) with 6 amu window.
• Fragmentation: EThcD (8 ms reaction, SA CE 15%), HCD (CE 25%), UVPD (25 pulses).

Main results and discussion

• Intact mass spectra resolved unmodified and mono- or di-phosphorylated VP2 proteoforms and a truncated VP3 clip.
• EThcD, HCD, and UVPD each provided complementary fragment ions, unambiguously localizing phosphosites at Ser-11 and Thr-56 in VP2.
• Sequence coverage for mono-phosphorylated VP2 improved by combining methods (up to ~50% residue cleavages versus 11–17% individually).
• VP3 clip sequence coverage increased from 13% (HCD alone) to 65% when combining all three dissociation techniques.

Benefits and practical applications

• Accurate localization of low-abundance PTMs on intact viral capsid proteins.
• Enhanced confidence in truncated proteoform identification.
• High sensitivity suitable for limited sample volumes of AAV preparations.
• Broad applicability to analytical and quality control workflows in biopharma.

Future trends and possibilities

Advances may include:

• Extension to other AAV serotypes and engineered capsids.
• Integration with automated data processing pipelines for high-throughput screening.
• Combination with native MS to study capsid assembly and host-protein interactions.
• Improved fragmentation strategies (e.g., hybrid UVPD-ETD) for further sequence coverage gains.

Conclusion

This study validates a targeted top-down LC-MS workflow using multiple fragmentation techniques on an Orbitrap Ascend Tribrid instrument for comprehensive PTM localization and proteoform sequence confirmation in AAV capsid proteins. Combining EThcD, HCD, and UVPD significantly boosts sequence coverage and analytical confidence, supporting rigorous characterization requirements in gene therapy development.

Reference

Reiko Kiyonami et al. Characterization of adeno-associated viral proteins proteoforms using a top-down MS approach on a UHPLC-Orbitrap Ascend Tribrid mass spectrometer. Thermo Fisher Scientific Application Note 002599.