In view of the significance of Asn deamidation and Asp
isomerization to isoAsp at certain sites for protein aging
and turnover, it was desirable to challenge the extreme
analytical power of electrospray tandem mass spectrometry
(ESI-MS/MS) for the possibility of a site-specific detection
of this posttranslational modification. For this purpose,
synthetic l-Asp/l-isoAsp containing oligopeptide
pairs were investigated by ESI-MS/MS and low-energy collision-induced
dissociation (CID). Replacement of l-Asp by l-isoAsp
resulted in the same kind of shifts for all 15 peptide pairs
investigated: (1) the b/y intensity ratio of complementary b and y
ions generated by cleavage of the (l-Asp/l-isoAsp)-X
bond and of the X-(l-Asp/l-isoAsp) bond
was decreased, and (2) the Asp immonium ion abundance at
m/z 88 was also decreased. It is proposed
that the isoAsp structure hampers the accepted mechanism
of b-ion formation on both its N- and C-terminal side.
The b/y ion intensity ratio and the relative immonium ion
intensity vary considerably, depending on the peptide sequence,
but the corresponding values are reproducible when recorded
on the same instrument under identical instrumental settings.
Thus, once the reference product ion spectra have been
documented for a pair of synthetic peptides containing
either l-Asp or l-isoAsp, these identify
one or the other form. Characterization and relative quantification
of l-Asp/l-isoAsp peptide mixtures are
also possible as demonstrated for two sequences for which
isoAsp formation has been described, namely myrG-D/isoD-AAAAK
(deamidated peptide 1–7 of protein kinase A catalytic
subunit) and VQ-D/isoD-GLR (deamidated peptide 41–46
of human procollagen alpha 1). Thus, the analytical procedures
described may be helpful for the identification of suspected
Asn deamidation and Asp isomerization sites in proteolytic
digests of proteins.
