An antibiotic linked to peptides and proteins is released by electron capture dissociation fourier transform ion cyclotron resonance mass spectrometry

Abstract

Desfuroylceftiofur (DFC) is a bioactive ␤-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins 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. 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 of2ϩ 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. eftiofur is a widely used broad-spectrum thirdgeneration cephalosporin antibiotic approved for use to treat infections in cattle, swine, sheep, goats, turkeys, and chickens. The four-membered ring structure of ␤-lactam antibiotics (which include penicillins and cephalosporins) is responsible for their antimicrobial activity against gram-positive and gram-negative bacteria by covalently binding to, and interrupting the function of, enzymes responsible for bacterial cell wall synthesis. Upon intramuscular injection, ceftiofur is rapidly metabolized (t 1/2 Ͻ 10 min) to desfuroylceftiofur (DFC) through hydrolytic cleavage of its thioester bond, generating furoic acid and a sulfhydryl moiety on DFC (Figure 1). Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC is bound to plasma and tissue proteins at cysteine residues via disulfide bonds [1,2]. It is estimated that 89% of DFC is covalently bound, through disulfide bonds, to plasma and tissue proteins. The remaining 11% is "free" in the form of the metabolite: Desfuroylceftiofur cysteine disulfide. DFC bound to amino acids, peptides, or proteins through disulfide bonds retains its antibacterial activity because its ␤-lactam ring remains intact.With the advent of electrospray ionization (ESI) [3] and matrix-assisted laser desorption/ionization (MALDI) [4,5] mass spectrometry, it has become relatively easy to desorb and ionize high molecular weight biomolecules into the gas phase. The higher charge states achievable with ESI lower the m/z of high molecular weight proteins to within the upper mass range (m/z 2000 -3000) of quadrupole, ion trap, and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. The number of positive charges (protons) that can attach to a protein depends on the number, location,

Evaluation and optimization of electron capture dissociation efficiency in fourier transform ion cyclotron resonance mass spectrometry

“…(J Am Soc Mass Spectrom 2005, 16, 1060 -1066) © 2005 American Society for Mass Spectrometry E lectron capture dissociation (ECD) [1,2] is a relatively new MS/MS technique that has become popular because it provides better peptide and protein sequence coverage compared with other dissociation methods, and retains labile post-translational modifications (e.g., glycosylation [3,4] and phosphorylation [5][6][7][8]. Recent applications also include fatty acids [9], antibiotic/protein complexes [10], ubiquitination [11], oligonucleotides [12], and histones [13][14][15]. Despite its proven analytical utility, ECD suffers from limited conversion efficiency of precursor to product ions.…”

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

“…Since the pharmacophorič -lactam ring in the thiol metabolite remains intact, the peptide-bound drug fraction retains its antibacterial activity and this fact can account for the long-lasting action of the drug in vivo. 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 […”

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

“…For instance, electron capture dissociation (ECD) mass spectrometry has demonstrated strong preference for dissociation of disulfide bonds in peptides and proteins 3–5. Very recently, ECD‐Fourier transform ion cyclotron resonance mass spectrometry was used to confirm that DFC binds to peptides and proteins through disulfide bonds 6,7. There have been numerous studies of the collision‐activated dissociation (CAD), post‐source and in‐source dissociation of disulfide‐linked peptides and proteins using a variety of ionization techniques and mass analyzers 8–16.…”