|Fagerquist, Clifton - Keith|
Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/17/2003
Publication Date: 3/10/2003
Citation: FAGERQUIST, C.K., HUDGINS, R.R., EMMETT, M.R., HAKANSSON, K., MARSHALL, A.G. A BIOACTIVE ANTIOBIOTIC LINKED TO PEPTIDES IS RELEASED BY ELECTRON CAPTURE DISSOCIATION FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY. JOURNAL OF AMERICAN SOCIETY FOR MASS SPECTROMETRY. 2003. Interpretive Summary: Ceftiofur is a widely used broad-spectrum beta-lactam antibiotic approved for use to treat infections in cattle, swine, sheep, goats, turkeys and chickens. Upon injection, ceftiofur is rapidly metabolized to desfuroylceftiofur (DFC) and retains the antimicrobial activity of the parent. Previous experiments tend to indicate that approximately 90% of DFC binds to plasma and tissue proteins. The nature of the binding is not known for certain, however DFC would retain its antimicrobial activity (even if it is bound to proteins), thus, it is important to detect protein-bound DFC as this constitutes an antibiotic residue. A promising technique for the detection of protein-bound DFC is electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). We have reacted DFC with two peptides to form DFC-peptide complexes, and then analyzed the products by ECD FT-ICR MS. Our results support the conclusion that DFC binds to proteins through disulfide bonds, which has implications for detection in edible tissue, and suggests ECD FT-ICR MS is a potentially useful technique for this purpose.
Technical Abstract: Desfuroylceftiofur (DFC) is a bioactive antibiotic metabolite which has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a thiol reducing agent, suggesting that DFC is bound to plasma proteins and tissue through disulfide bonds. We have reacted DFC with [Arg-8]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds in the gas phase. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H) + 2H]2+ from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H]4+ shows preferential dissociative loss of DFC. Combined with HPLC, ECD-FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.