An antibiotic linked to peptides and proteins is released by electron capture dissociation fourier transform ion cyclotron resonance mass spectrometry.
Desfuroylceftiofur (DFC) is a bioactive beta-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 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.
Fagerquist
Clifton K
CK
United States Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038, USA. kfagerquist@arserrc.gov
Hudgins
Robert R
RR
Emmett
Mark R
MR
Håkansson
Kristina
K
Marshall
Alan G
AG
D000900:Anti-Bacterial Agents / Q000737:chemistry*; D002511:Cephalosporins / Q000737:chemistry*; D005583:Fourier Analysis; D007328:Insulin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D014667:Vasopressins / Q000737:chemistry
D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D007328:Insulin; D010455:Peptides; D011506:Proteins; D014667:Vasopressins; C053503:ceftiofur