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 O-18 water to monitor succinimide hydrolysis. O-18-labeled isoaspartic acid and aspartic acid peptides were produced by hydrolysis of a succinimide-containing protein in O-18 water ((H2O)-O-18) followed by tryptic digestion in regular water ((H2O)-O-16). The peptides that had O-18 incorporated were 2 Da heavier than their O-16 native counterparts. The mass difference was detected and quantified by electrospray time-of-flight mass spectrometry. The amount of O-18 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 the site of aspartic acid isomerization. At this site, there were 20% isoaspartic acid and 80% aspartic acid detected by peptide mapping in the degraded sample (8 weeks, 45 degrees C, pH 5.0). Hydrolysis in O-18 water showed that 80% of the isoaspartic acid and 6% of the aspartic acid had O-18 incorporated. The only explanation of O-18 incorporation was the presence of succinimide in the sample. Together, a total of 21% (0.8 x 20% isoaspartic acid + 0.06 x 80% aspartic acid) of aspartic acid residue 30 was found to be present in the form of succinimide in this degraded sample. As a control, the same sample, analyzed using regular O-16 water did not show any incorporation of O-18 water. By monitoring the amount of O-18-labeled isoaspartic acid and aspartic acid over time under both denaturing and native conditions at pH 8.2, we found that, at denaturing conditions, succinimide at light chain residue 30 hydrolyzed very rapidly (in less than 5 s), but slower (succinimide half-life of similar to 6 h) under native conditions. We also found that, under denaturing conditions, succinimide hydrolyzed at an isoaspartic acid/aspartic acid ratio of 3.5:1, but hydrolyzed almost exclusively to aspartic acid under native conditions. This finding indicates that protein structure plays an important role in the kinetics of succinimide hydrolysis as well as in the generation of the hydrolysis products isoaspartic acid and aspartic acid.