|Fagerquist, Clifton - Keith|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2002
Publication Date: 9/1/2002
Citation: FAGERQUIST, C.K., HUDGINS, R.R., EMMETT, M.R., HAKANSSON, K., MARSHALL, A.G. SELECTIVE CLEAVAGE OF A SMALL ANTIBIOTIC DRUG MOLECULE FROM A PEPTIDE AND PROTEIN IN THE GAS PHASE USING ELECTRON CAPTURE DISSOCIATION AND FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY. MEETING ABSTRACT. 2002.
Technical Abstract: Ceftiofur is a widely used third generation beta-lactam antibiotic approved for use in cattle, swine and chickens for treatment of respiratory infections. Upon injection, ceftiofur is rapidly metabolized to its bioactive metabolite: desfuroylceftiofur (DFC) which has a free thiol group. Previous experiments have shown release of DFC from plasma extracts sand tissue after addition of a thiol reducing agent suggesting that DFC is bound to plasma proteins and tissues via disulfide bonds. In order to test this hypothesis, we have reacted DFC with the peptide vasopressin and the protein bovine pancreatic insulin both of which have disulfide bonds. The reaction products were ionized by electrospray ionization and analyzed using electron capture dissociation (ECD) and Fourier transform ion cyclotron resonance mass spectrometry. ECD has previously shown strong preference for cleavage of disulfide bonds. We observed attachment of one and two molecules of DFC to vasopressin consistent with it having two cysteine residues. The + 2 charge state of the vasopressin-DFC(2) complex was isolated by Stored Waveform Inverse Fourier Transform (SWIFT) and analyzed by ECD. ECD shows a peak corresponding to dissociative loss of a DFC radical as well as a peak corresponding to combined loss of a DFC radical and a DFC molecule. The latter suggests a step-wise sequential loss with reformation of the original cyclic structure of the peptide. For bovine pancreatic insulin, we observed attachment of one, two and three DFC molecules. The + 4 charge state of the insulin-DFC complex was SWIFT isolated and analyzed by ECD. We observed dissociative loss of DFC from the +3 and +2 charge states. These results strongly support the attachment of DFC to peptides and proteins through disulfide bonds.