Characterization of a β-Asp33 Isoform of Recombinant Hirudin Sequence Variant 1 by Low-energy Collision-induced Dissociation
✍ Scribed by Schindler, Patrick; Müller, Dieter; Märki, Walter; Grossenbacher, Hugo; Richter, Wilhelm J.
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 683 KB
- Volume
- 31
- Category
- Article
- ISSN
- 1076-5174
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✦ Synopsis
A new low-concentration congener (Ib) of recombinant hirudin sequence variant 1 was structurally characterized as a &Asp33 isoform of the parent protein (Ia). a-P Isomerization a t the 33-position was expected in view of the previous isolation of a potential precursor (Asp33-Gly34-anhydro-hirudin (lc)), i.e. a succinimide-type dehydration product liable to undergo facile hydrolysis with ring opening, yielding p-(along with a-) aspartates. In order to identify and locate the modified site in Ib, a sufficiently small peptide (I28-3Sj-octapeptide Illb) 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 [ b, + H,0If ion indicative of p-Asp in the neighboring 'n + 1' position was observed for n = 5. This located the p-Asp residue unambiguously in the 4-position of IIIb and thus, as expected, in the 33-position of Ib. The formation of this highly diagnostic [b, + H,O] + 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 '80-labeling of the suspected migratory oxygen atom (fl-Asp33-CO ' 'OH) together with the low-energy genesis suggest a specific chargetriggered 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.