Collision‐activated cleavage of a peptide/antibiotic disulfide linkage: possible evidence for intramolecular disulfide bond rearrangement upon collisional activation

Abstract

Ceftiofur is an important veterinary beta-lactam antibiotic whose bioactive metabolite, desfuroylceftiofur, has a free thiol group. Desfuroylceftiofur (DFC) was reacted with two peptides, [Arg8]-vasopressin and reduced glutathione, both of which have cysteine residues to form disulfide-linked peptide/antibiotic complexes. The products of the reaction, [vasopressin + (DFC-H) + (DFC-H) + H]+, [(vasopressin+H) + (DFC-H) + H]+ and [(glutathione-H) + (DFC-H) + H]+, were analyzed using collision-activated dissociation (CAD) with a quadrupole ion trap tandem mass spectrometer. MS/MS of [vasopressin + (DFC-H) + (DFC-H) + H]+ resulted in facile dissociative loss of one and two covalently bound DFC moieties. Loss of one DFC resulted from either homolytic or heterolytic dissociation of the peptide/antibiotic disulfide bond with equal or unequal partitioning of the two sulfur atoms between the fragment ion and neutral loss. Hydrogen migration preceded heterolytic dissociation. Loss of two DFC moieties from [vasopressin + (DFC-H) + (DFC-H) + H]+ appears to result from collision-activated intramolecular disulfide bond rearrangement (IDBR) to produce cyclic [vasopressin + H]+ (at m/z 1084) as well as other cyclic fragment ions at m/z 1084 +/- 32 and +64. The cyclic structure of these ions could only be inferred as MS/MS may result in rearrangement to non-cyclic structures prior to dissociative loss. IDBR was also detected from MS(3) experiments of [vasopressin + (DFC-H) + (DFC-H) + H]+ fragment ions. MS/MS of [(glutathione-H) + (DFC-H) + H]+ resulted in cleavage of the peptide backbone with retention of the DFC moiety as well as heterolytic cleavage of the peptide/antibiotic disulfide bond to produce the fragment ion: [(DFC-2H) + H]+. These results demonstrate the facile dissociative loss by CAD of DFC moieties covalently attached to peptides through disulfide bonds. Published in 2004 by John Wiley & Sons, Ltd.

Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

“…Disulfide scrambling may be explained by the mechanism shown in Figure 3a. The observed fragment ion IV is the consequence of photoinitiated formation of four sulfhydryl radicals and rapid intramolecular recombination, a nonreductive process consistent with previously published results from CAD experiments [14].…”

“…Fragmentation across the S-S-bond (homolytic and heterolytic) provides evidence for the structure [12,13]. Among other feasible fragmentation mechanisms, one paper has reported evidence for disulfide scrambling in the gas phase during CAD [14]. A free thiol-containing antibiotic metabolite, desfuroylceftiofur (DFC), was used to derivatize peptides into disulfide-linked bioactive compounds.…”

Characterization of the disulfides of bio‐thiols by electrospray ionization and triple‐quadrupole tandem mass spectrometry

“…At the investigated value of the collision energy, the [MH H 2 S] C fragment at m/z 130 in the spectrum of protonated N-acetylcysteine (14) is not observable (<<1%), whereas that at m/z 158 in that of protonated N-acetylpenicillamine (18) is observable, although at very low abundance, thus confirming that loss of H 2 S to yield the protonated oxazoline is a strongly disfavoured process. This conclusion may in turn explain why the formation of the corresponding a-type fragments in the spectra of the disulfides is a minor process, when compared with heterolytic fragmentation of the disulfide bond.…”

“…Although the unimolecular decomposition of protonated amino acids has been thoroughly studied in the past, in both experimental and computational studies, 19,23 -25 the spectra of some thiolamino acids and N-acetylamino acids (homocysteine (8), penicillamine (10), N-acetylcysteine (14) and N-acetylpenicillamine (18)) employed as starting reagents to prepare the examined disulfides have not been reported and are therefore described here, since some of their unimolecular decomposition processes have been employed to interpret those of the corresponding disulfides.…”

“…12 The mixed disulfides of desfuroyl-ceftiofur with cysteine-or cystine-containing peptides, such as Arg 8 -vasopressin (VPS 2 ), glutathione and bovine insulin, have recently been characterized both by collisionally activated dissociation (CAD) in an ion trap 13 and by electroncapture decomposition in a Fourier transform ion cyclotron resonance (FTICR) instrument. 14 Identification of thiolated proteins has often employed non-mass spectrometric techniques, such as the spectrophotometric assay of free thiol groups with Ellmann's reagent, radioactive labeling with […”