Rapid Highly-Efficient Digestion and Peptide Mapping of Adeno-Associated Viruses

Adeno-associated
viruses (AAVs) comprise an area of rapidly growing
interest due to their ability to act as a gene delivery vehicle in
novel gene therapy strategies and vaccine development. Peptide mapping
is a common technique in the biopharmaceutical industry to confirm
the correct sequence, product purity, post-translational modifications
(PTMs), and stability. However, conventional peptide mapping is time-consuming
and has proven difficult to reproduce with viral capsids because of
their high structural stability and the suboptimal localization of
trypsin cleavage sites in the AAV protein sequences. In this study,
we present an optimized peptide mapping-based workflow that provides
thorough characterization within 1 day. This workflow is also highly
reproducible due to its simplicity having very few steps and is easy
to perform proteolytic digestion utilizing thermally stable pepsin,
which is active at 70 °C in acidic conditions. The acidic conditions
of the peptic digestions drive viral capsid denaturation and improve
cleavage site accessibility. We characterized the efficiency and ease
of digestion through peptide mapping of the AAV2 viral capsid protein.
Using nanoflow liquid chromatography coupled with tandem mass spectrometry,
we achieved 100% sequence coverage of the low-abundance VP1 capsid
protein with a digestion process taking only 10 min to prepare and
45 min to complete the digestion.