An 18O-labeling assisted LC/MS method for assignment of aspartyl/isoaspartyl products from Asn deamidation and Asp isomerization in proteins.

An (18)O-labeling assisted LC/MS method was designed for unambiguous assignment of aspartyl/isoaspartyl products produced by Asn deamidation and Asp isomerization. By preparing the acid- and base-catalyzed deamidation standards in H2(18)O, isomer-specific mass tags were introduced to aspartyl- and isoaspartyl-containing peptides, which could be easily distinguished by mass spectrometry (MS). In contrast to the traditional ways of assigning the isomers on the basis of their elution order in reverse phase HPLC, the new method is more reliable and universal. Furthermore, the new method can be applied to the entire protein digest, and is therefore more time- and cost-effective compared with existing methods that use synthetic aspartyl- and isoaspartyl-containing peptide standards. Finally, since the identification of isomers in the new method only relies on LC/MS analysis, it can be easily implemented using the most basic and inexpensive MS instrumentation, thus providing an attractive alternative to tandem MS based approaches. The feasibility of this new method is demonstrated using a model peptide as well as the entire digest of human serum transferrin.