18O Labeling Method for Identification and Quantification of Succinimide in Proteins

We have developed a new method for identification and quantification of succinimide in proteins. The method utilizes 18O water to monitor succinimide hydrolysis. 18O-labeled isoaspartic acid and aspartic acid peptides were produced by hydrolysis of a succinimide-containing protein in 18O water (H218O) followed by tryptic digestion in regular water (H216O). The peptides that had 18O incorporated were 2 Da heavier than their 16O native counterparts. The mass difference was detected and quantified by electrospray time-of-flight mass spectrometry. The amount of 18O incorporation into the isoaspartic acid- and aspartic acid-containing peptides was used to quantify the amount of succinimide present in the native sample. The method was applied to analyze a degraded recombinant monoclonal antibody, which exhibited the accumulation of succinimide after storage in mildly acidic buffers at elevated temperatures for a few weeks. We unambiguously identified amino acid residue 30 located in the antibody light chain as ...