HOMEPRODUCTSCOMPANYCONTACTFAQResearchDictionaryPharmaSign Up FREE or Login

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.
AuthorsP Schindler, D Müller, W Märki, H Grossenbacher, W J Richter
JournalJournal of mass spectrometry : JMS (J Mass Spectrom) Vol. 31 Issue 9 Pg. 967-74 (Sep 1996) ISSN: 1076-5174 [Print] England
PMID8831150 (Publication Type: Journal Article)
Chemical References
  • Antithrombins
  • Disulfides
  • Hirudins
  • Recombinant Proteins
  • Trypsin
Topics
  • Alkylation
  • Amino Acid Sequence
  • Antithrombins (chemistry)
  • Disulfides (chemistry)
  • Hirudins (chemistry)
  • Hydrolysis
  • Isomerism
  • Mass Spectrometry
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Recombinant Proteins (chemistry)
  • Saccharomyces cerevisiae (genetics, metabolism)
  • Spectrophotometry, Ultraviolet
  • Trypsin

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research graph!


Choose Username:
Email:
Password:
Verify Password:
Enter Code Shown: