AccuMAP® Low pH Protein Digestion Kit

Trypsin and other proteases commonly used in peptide mapping sample preparation favor alkaline pH in order to efficiently digest proteins. To avoid artificial nonenzymatic PTMs induced at these conditions, we developed trypsin digestion at low pH. To restore trypsin cleavage efficiency at lysine sites we supplemented trypsin with a special, low pH-resistant recombinant Lys-C (rLys-C) protease. By supplementing trypsin with low pH resistant rLys-C we achieved efficient tryptic digestion at low pH.

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.