Sample Preparation Matters for Peptide Mapping to Evaluate Deamidation of Adeno-Associated Virus Capsid Proteins Using Liquid Chromatography-Tandem Mass Spectrometry.

Adeno-associated viral capsid proteins (AAV VP) are the major components that determine the tissue specificity and immunogenicity, and transduction performance of the vector. It was reported that asparagine deamidation of AAV capsid proteins leads to charge variants/heterogeneity and altered vector function, reduction of stability, and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins and is mostly detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based peptide mapping. However, deamidation can be spontaneously introduced during sample preparation before LC-MS/MS analysis. So far, no optimal sample preparations, instead, traditional sample preparation has been used for AAV VP peptide mapping, resulting in exaggerating the original deamidation levels. It is important to accurately monitor and provide true value of asparagine deamidation for development of AAV gene therapy products. In this study, we evaluated denaturation temperatures, digestion durations, and digestion temperatures using three different sample preparation formats for LC-MS/MS-based assessment of deamidation of AAV9 capsid proteins. The results demonstrated that the optimal sample preparation method for AAV9 VP peptide mapping minimized asparagine deamidation artifacts. Although AAV9 was used for method optimization, this study may also provide a guidance on how to control deamidation artifacts for other AAV serotypes.