Characterization of asparagine deamidation and aspartic acid isomerization is an important aspect of biotherapeutic protein analysis. Succinimide has long been known to be an intermediate product, but its analysis remains challenging due to its instability. We have developed a paradigm in which two interlinked analytical methods are used to develop an optimized approach to analyze succinimide. In the first method, low-pH protein digestion is used for detailed characterization of succinimide with peptide mapping. At low pH, succinimide is stable and can be analyzed with accurate mass measurements and tandem mass spectrometry to confirm its identity and localize its modification site.
Characterization and Quantification of Succinimide Using Peptide Mapping Under Low-pH Conditions and Hydrophobic Interaction Chromatography (2019) Anal. Biochem. 566, 151–59.