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Characterization of a β‐Asp33 Isoform of Recombinant Hirudin Sequence Variant 1 by Low‐energy Collision‐induced Dissociation
Author(s) -
Schindler Patrick,
Müller Dieter,
Märki Walter,
Grossenbacher Hugo,
Richter Wilhelm J.
Publication year - 1996
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/(sici)1096-9888(199609)31:9<967::aid-jms381>3.0.co;2-k
Subject(s) - chemistry , electrospray ionization , stereochemistry , tandem mass spectrometry , peptide , peptide sequence , dissociation (chemistry) , cysteine , mass spectrometry , enzyme , biochemistry , chromatography , organic chemistry , gene
A new low‐concentration congener (Ib) of recombinant hirudin sequence variant 1 was structurally characterized as a β‐Asp33 isoform of the parent protein (Ia). α‐β 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 β‐ (along with α‐) 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 fol‐lowed 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 n +H 2 O] + ion indicative of β‐Asp in the neighboring ‘ n +1’ position was observed for n = 5. This located the β‐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 [b n +H 2 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 18 O‐labeling of the suspected migratory oxygen atom (β‐Asp33–CO 18 OH) 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.