Mass spectrometry analysis of post translational modification in adeno-associated virus capsid proteins

Introduction:

Adeno-associated virus (AAV) capsids are currently the most favoured vectors for gene delivery. Recombinant viruses produced in a specific cell line are likely to acquire unique post-translational modifications during the intracellular maturation of their capsid proteins. These post-translational modifications (PTM) on the capsid proteins are known to regulate the stability, packaging and transduction efficiency and shield potentially immunogenic protein epitopes. Understanding these fine tune regulations may aid in gene therapy development.

Methodology:

AAV vectors were produced by triple transfection in HEK293T cells. The cell lysate and supernatant were extracted and purified using cesium chloride method and affinity chromatography respectively post 72 hours. The purified protein was denatured and alkylated. Before trypsin digestion, EndoH treatment was given to enrich the glyco proteins. TripleTOF6600 coupled with UPLC was used for protein identification. The molecular dynamics simulation approach was used to assess the protein structure and stability upon modification.

Results:

Our approach identified comparatively a greater number of viral capsid proteins in affinity chromatography purified supernatants than cell lysates. Moreover, we found lesser host cell protein carry over contamination in affinity purified samples. A total of 4, 12 and 3 novel glycosylation modifications and 8, 14 and 3 phosphorylation modifications were identified for AAV6, AAV8 and AAV9 vectors respectively. Interestingly, our MDS results showed that PTMs on loop 4, loop 6 and loop 8 region of the capsid proteins have functional importance in the capsid proteins.

Conclusion:

The current data reported novel capsid modifications which can be explored further to understand the biological relevance of modified AAV in transduction, stability and immune evasion. This will aid in development of novel bioengineered AAV vectors for gene therapy applications.

Keywords:

Adeno-associated virus; capsid; proteomics; LC-MS identification; PTM; Molecular Dynamics simulation