A new low-concentration congener (Ib) of recombinant
hirudin sequence variation 1 was structurally characterized as a beta-Asp33
isoform of the parent
protein (Ia). alpha-beta Isomerization at the 33-position was expected in view of the previous isolation of a potential precursor (Asp33-Gly34-anhydro-hirudin (Ic)), i.e., a
succinimide-type
dehydration product liable to undergo facile hydrolysis with ring opening, yielding beta- (along with alpha-) aspartates. In order to identify and locate the modified site in Ib, a sufficiently small
peptide ([28-35]-octapeptide IIIb) was prepared by
disulfide bond reduction, S-alkylation (pyridylethylation) and twofold enzymatic degradation (
Glu-C protease followed by
trypsin). When [M + H] +
ions of IIIb were analyzed by electrospray ionization tandem mass spectrometry (ESIMS/MS) and low-energy collision-induced dissociation (CID), a singular [bn + H2O]+ ion indicative of beta-Asp in the neighboring 'n + 1' position was observed for n = 5. This located the beta-Asp residue unambiguously in the 6-position of IIIb and thus, as expected, in the 33-position of Ib. The formation of this highly diagnostic [bn + H2O]+ ion, for which precedents had only been reported for CID under high-energy conditions, requires net
OH migration from one to another
amino acid position. Confirmatory results from 18O-labeling of the suspected migratory
oxygen atom (beta-Asp33-CO18OH) together with the low-energy genesis suggest a specific charge-triggered rather than charge-remote mechanism for the formation of the ion. The analogy of this process to the ejection of the C-terminal
amino acid similarly involving net
OH rearrangement is discussed.