Zhou, Yu and Wang, Yueju (2022) Sample Preparation Matters for Peptide Mapping to Evaluate Deamidation of Adeno-Associated Virus Capsid Proteins Using Liquid Chromatography–Tandem Mass Spectrometry. Human Gene Therapy, 33 (15 and). pp. 821-828.

Abstract

Adeno-associated viral capsid proteins (AAV VP) are the major components that determine the tissue specificity and
immunogenicity, and in vivo 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.

Item Type:	Article
Keywords:	AAV, capsid proteins, deamidation, peptide mapping, sample preparation
Date Deposited:	09 Sep 2022 00:45
Last Modified:	09 Sep 2022 00:45
URI:	https://oak.novartis.com/id/eprint/45362