Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis.

Nonenzymatic asparagine (Asn) deamidation is one of the commonly observed posttranslational modifications of proteins. Recent development of several specific analytical methods has allowed for efficient identification and differentiation of the deamidation products (i.e., isoaspartate [isoAsp] and aspartate [Asp]). Isotope labeling of isoAsp and Asp that are generated during sample preparation by 18O has been developed and can differentiate isoAsp and Asp as analytical artifacts from those present in the samples prior to sample preparation for an accurate quantitation. However, the 18O labeling procedure has a limitation due to the additional incorporation of up to two 18O atoms into the peptide C-terminal carboxyl groups. Variability in the incorporation of 18O atoms into the peptide C-terminal carboxyl groups results in complicated mass spectra and hinders data interpretation. This limitation can be overcome by the dissection of the complicated mass spectra using a calculation method presented in the current study. The multiple-step calculation procedure has been successfully employed to determine the levels of isoAsp and Asp that are present in the sample prior to sample treatment.