An <sup>18</sup>O‑Labeling Assisted LC/MS Method
for Assignment of Aspartyl/Isoaspartyl Products from Asn Deamidation
and Asp Isomerization in Proteins
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Abstract
An <sup>18</sup>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 H<sub>2</sub><sup>18</sup>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