(M1130-02-08) Comprehensive Characterization of Adeno-Associated Virus Capsid Proteins via Peptide Mapping and Post-Translational Modification Analysis

Purpose: Adeno-associated virus (AAV) stands as a prominent vector in gene therapy, especially for monogenic diseases, due to its high transduction efficiency, low immunogenicity, and sustained therapeutic effects. Its significant potential is underscored by the six FDA-approved AAV-based therapies and over 120 ongoing clinical trials. The icosahedral capsid of AAV is composed of three viral proteins—VP1, VP2, and VP3—present in a defined stoichiometry. While serotype-specific variations are well-documented, further investigation into post-translational modifications (PTMs) is essential for a deeper understanding of their influence on viral infectivity and stability. Comprehensive characterization of these viral proteins is therefore critical for advancing AAV-mediated therapies and ensuring the quality of the final product. Liquid chromatography-mass spectrometry (LC/MS)-based peptide mapping and PTM analysis provide an efficient and accurate workflow for evaluating the quality attributes of AAV capsid proteins, directly impacting the safety and efficacy of these gene therapies.

Methods: The AAV8 reference material had a total volume of 20 µL at a concentration of 2 × 10¹³ vg/mL, corresponding to an estimated protein concentration of 0.12 µg/µL and a total of 2.4 µg protein for digestion. The sample was denatured in 6 M urea and 1 mM DTT at 80 °C for 30 minutes, then alkylated with 15 mM iodoacetamide for 30 minutes at room temperature in the dark. Following alkylation, the sample was cooled to room temperature and diluted threefold with buffer (50 mM Tris-HCl, 1 mM CaCl₂, pH 7.5), reducing the urea concentration to below 2 M. Proteolytic digestion was carried out overnight at 37 °C using 0.4 µg of trypsin or chymotrypsin. Digestion was quenched by adding formic acid to a final concentration of 10%, and the sample was directly injected into a Q-TOF mass spectrometer for analysis. Chromatographic separation was performed on a C18 column (150 mm × 2.1 mm, 2.7 µm) at a flow rate of 0.2 mL/min, using water and acetonitrile containing 0.1% formic acid as mobile phases. Data were acquired in data-dependent acquisition (DDA) mode.

Results: Trypsin cleaves peptides at the C-terminus of lysine (K) and arginine (R), while chymotrypsin targets the C-terminus of aromatic residues, including tyrosine (Y), phenylalanine (F), and tryptophan (W). Due to regions in AAV8 capsid proteins that are highly enriched in lysine and arginine, achieving complete sequence coverage using trypsin alone is challenging. To address this, chymotrypsin was incorporated to complement tryptic digestion and enhance coverage. Both enzymes effectively digested the AAV8 capsid proteins, generating chromatograms with an increased number of peptide peaks. All matched peptides exhibited mass errors below 6 ppm and were confirmed by at least one corresponding MS/MS spectrum. Carbamidomethylation (C) was set as a fixed modification, while variable modifications included oxidation (M), deamidation (N/Q), pyro-glutamate formation (N-terminal Q), ammonia loss, acetylation (protein N-terminus), and phosphorylation (S/T/Y). Most identified peptides demonstrated excellent mass accuracy, with mass errors under 1 ppm. The VP3 protein sequence is fully nested within VP2, which in turn is nested within VP1. Sequence coverage was 96.1% for VP1, 97.5% for VP2, and 100% for VP3. Several post-translational modifications (PTMs) were detected, including deamidation, oxidation, acetylation, phosphorylation, and pyro-glutamate formation. These PTMs can influence AAV vector function, potentially enhancing or impairing transduction efficiency. For example, the peptide TWALPTYNNHLYK (VP1 247–259) displayed deamidation at an asparagine residue, with both native and modified forms observed. A mass shift of +0.98 Da in the deamidated peptide, especially evident in the y6–y9 fragment ions, enabled clear differentiation from the unmodified form. Similarly, the peptide SSFYCLEYFPSQMLR (VP1 393–407) exhibited a mass shift of +15.99 Da in the y6–y9 fragment ions, consistent with methionine oxidation.

Conclusion: A comprehensive workflow, spanning sample preparation to LC-MS analysis, was established for the precise characterization of AAV capsid protein sequences. Proteolytic digestion using trypsin and chymotrypsin achieved near-complete sequence coverage, while high-quality MS/MS spectra enabled confident peptide identification and accurate localization of post-translational modifications.


Sequence coverage map of AAV8 capsid proteins (VP1, VP2, and VP3) using different enzymatic digests. Blue: trypsin. Magenta: chymotrypsin.