COLLISION-ACTIVATED CLEAVAGE OF A PEPTIDE/ANTIBIOTIC DISULFIDE LINKAGE: EVIDENCE OF INTRAMOLECULAR DISULFIDE BOND REARRANGEMENT UPON COLLISIONAL ACTIVATION

Authors: Fagerquist, Clifton - Keith

Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2004
Publication Date: 3/30/2004
Citation: FAGERQUIST, C.K. COLLISION-ACTIVATED CLEAVAGE OF A PEPTIDE/ANTIBIOTIC DISULFIDE LINKAGE: EVIDENCE OF INTRAMOLECULAR DISULFIDE BOND REARRANGEMENT UPON COLLISIONAL ACTIVATION. RAPID COMMUNICATIONS IN MASS SPECTROMETRY.

Interpretive Summary: The screening for drug residues in edible tissue is important in order to protect public health. The screening often involves detection, not of the original drug, but a metabolite that may bind to large biomolecules. Ceftifur is a widely used veterinary antibiotic approved for use in livestock. Its metabolite, desfuroylceftiofur (DFC), can bind to peptides and proteins found in edible tissue. Protein-bound DFC retains the antimicrobial activity of the parent compound and therefore constitutes a drug residue. To develop a screening method for detection of peptide-bound (or protein-bound DFC, we have reacted DFC with two peptides: vasopressin and glutathione and analyzed the reaction products using collision-activated dissociation tandem ion trap mass spectrometry (CAD/MSn). The results indicate that application of CAD/MSn can detect the presence of peptide- bound DFC. This information is useful for food safety regulatory agencies to develop appropriate methods for screening for ceftiofur in food matrices.

Technical Abstract: Desfuroylceftiofur, an important antibiotic metabolite, was reacted with two peptides [arg8]-vasopressin, a cyclic peptide having two cysteine residues, and reduced glutathione that has one cysteine residue. [Vasopressin+(DFC-H) + (DFC-H)+H]+, Vasopressin+(DFC-H)+ and [glutophione-H) + (DFC-H)+H] + were analyzed using collision 'activated dislocation quadruple ion trap mass spectrometry (CAD/MSn). MS/MS of [vasopressin+(DFC-H) + (DFC-H)+H]+ resulted in dislocative loss of two (DFC-H) to produce a fragment ion at m/z 1084. MS/MS/MS of the fragment ion at m/z 1084 confirms that it is native cyclic structure of vasopressin suggesting that the dissociative loss of two (DFC-H) involves an intramolecular disulfeide bond rearrangement in the gas phase. The presence of other fragment ions eliminate the possibility that [vasopressin+(DFC-H)+(DFC-)+H is a non-covalently bound complex of vasopressin and (DFC-H) dimmer. MS/MS of [(glutathione-H) + (DFC-H)+ resulted in cleavage of peptide backbone with retention of the DFC adduct as well as dissociative loss of the peptide to produce the fragment ion [(DFC-2H)+H]+. The formation of [(DFC-2H)+H+ appears to result from hydrogen migration from DFC adduct followed by rupture of the DFC-peptide disulfidebond. Dissociative loss of (DFC-2H) is also observed in MS/MS spectrum of [vasopressin+(DFC-H)+H]+. These results demonstrate the facile loss of DFC adducts covalently attached to peptides through disulfide bonds and thus detection of DFC/peptides conjugates by purely mass spectrometric techniques.