title: Characterization of a beta-Asp33 isoform of recombinant hirudin sequence variant 1 by low-energy collision-induced dissociation.
abstract: 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.
authors.affiliation: Central Research Services, Ciba-Geigy Ltd, Basle, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Schindler
authors.forename: D
authors.initials: D
authors.lastname: Müller
authors.forename: W
authors.initials: W
authors.lastname: Märki
authors.forename: H
authors.initials: H
authors.lastname: Grossenbacher
authors.forename: W J
authors.initials: WJ
authors.lastname: Richter
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D000991:Antithrombins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D006629:Hirudins / Q000737:chemistry*; D006868:Hydrolysis; D007536:Isomerism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
chemical_list: D000991:Antithrombins; D004220:Disulfides; D006629:Hirudins; D011994:Recombinant Proteins; D014357:Trypsin