Identification of leucine‐enkephalin radical oxidation products by liquid chromatography tandem mass spectrometry

Abstract

The oxidation of the peptide leucine-enkephalin (YGGFL) induced by the hydroxyl radical (HO*), formed under Fenton-like conditions [Cu (II)/H(2)O(2)], was studied and monitored by LC-MS. The oxidation products identified included products resultant from (a) the insertion of oxygen atoms (1-5), (b) peptide backbone cleavage (short-chain products formed by diamide pathway) and (c) radical-radical crosslinking reactions. In order to identify the modified residues, LC-MS/MS spectra were obtained. The insertion of oxygen atoms into the peptide originated hydroxide, di-hydroxide and/or hydroperoxide derivatives. In addition it was found that the aromatic amino acids are most susceptible to being hydroxylated, while the aliphatic amino acids are more prone to forming hydroperoxides. Oxidation products with double bonds were also identified. The short chain products resulted from the alpha-carbon radical of terminal amino acids (Tyr and Leu). Products resulting from cross-linking reactions between intact carbon-centered peptide radical (with and without one HO group) and a side chain radical (*C(7)H(7)O) were identified. It was found that, although all amino acids residues of the peptide undergo modifications, the N-terminal seems to be prone to oxidative modifications under these conditions.

The Chemistry of Protein Oxidation in Food

“…Cleavage of the C α −CO bond of backbone alkoxyl radicals ( 74 ) leads to an imide ( 75 ) and a peptide isocyanate ( 76 ) (diamide pathway, Scheme ). The respective reaction products ( 75 and 76 ) have only been characterized on the basis of MS/MS fragmentation spectra, even though the diamide pathway is energetically favored over α‐amidation . Backbone cleavage by metal‐catalyzed oxidation was reported to proceed through the diamide pathway preferentially adjacent to lysine or arginine side chains .…”

Peptide and protein drugs: The study of their metabolism and catabolism by mass spectrometry

“…Conventional methods to monitor oxidative modifications and damage of proteins have been UV, fluorescence, gel electrophoresis, and isoelectric focusing; however these methodologies provide limited structural information (Schey & Finley, 2000; Watson, Atsriku, & Oliveira, 2003). In a recent report, the identification and structural characterization of leucine‐enkephalin (YGGFL) radical oxidation products was carried out by LC‐MS/MS (Fonseca et al, 2008). Leucine‐enkephalin is a bioactive opioid peptide that serves as a morphine‐acting analgesic.…”

Analysis of oxidation process of cholecystokinin octapeptide with reactive oxygen species by high‐performance liquid chromatography and subsequent electrospray ionization mass spectrometry

“…Mass spectrometry (MS), particularly electrospray ionization mass spectrometry (ESI-MS), is useful in this regard because it enables the analysis of reactions involving changes in the molecular mass of peptides and proteins, such as glycosylation, phosphorylation, and redox reactions. Identification of specific residues of protein and peptides which were modified by ROS has been reported (Sharp et al, 2003;Fonseca et al, 2008). This study describes the usefulness of reversed-phase high-performance liquid chromatography (RP-HPLC), ESI-MS and MS/MS for timecourse change and characterization of the oxidation products of CCK8 by ROS in vitro.…”