Identification of oxidation products and free radicals of tryptophan by mass spectrometry

Abstract

New oxidation products and free radicals derived from tryptophan (Trp) oxidation under Fenton reaction conditions were identified using mass spectrometry. After the oxidation of tryptophan using hydrogen peroxide and iron (II) system (Fenton reaction), mono-and dihydoxy tryptophans and N-formylkynurenine were identified using electrospray mass spectrometry (ES-MS) and ES-MS/MS. Besides these products, new products resulting from the reaction of tryptophan and oxidized tryptophan and 3-methyl indole derivatives were also identified. The 3-methyl indole derivatives resulted, most probably, from the oxidation process and not from in-source processes. A dimer formed by cross-linking between two Trp radicals (Trp-Trp), similar to the previously described tyrosine dimer was observed, as well as the corresponding monohydroxy-dimer (Trp-Trp-OH). Tandem mass spectrometry was used to identify the structures of these new oxidation products. Free radicals derived from tryptophan oxidation under Fenton reaction were detected using as spin trap the DMPO. The free radical species originated during the oxidation reaction formed stable adducts with the spin trap, and these adducts were identified by ES-MS. New adducts of oxidized tryptophan radicals, namely monohydroxy-tryptophan and dihydroxy-Trp dimer radicals, with one and two DMPO spin trap molecules where identified. Tandem mass spectrometry was used to confirm the proposed structure of the observed adducts. (J

Metal-independent decomposition of hydroperoxides by halogenated quinones: Detection and identification of a quinone ketoxy radical

“…Along the same line, DMPO/CBQ-OH adduct may exist in 2 of the 3 possible redox forms: the DMPO/CBQ-OH nitroxide form and its corresponding nitrone form as shown in Scheme 3. Several previous studies showed that DMPO radical adducts were detected by MS in one or all of the 3 different forms (12)(13)(14)(15). Under our HPLC/ESI-MS experimental conditions and based on the FTICR high resolution MS data, the measured molecule ion peak at m/z 268.0383 could be assigned to the oxidized nitrone form (theoretical mass 268.0377) of the DMPO/CBQ-OH nitroxide radical adduct.…”

Characterization of Free Radical Spin Adducts of 5-Diisopropyloxy-Phosphoryl-5-Methyl-1-Pyrroline-N-Oxide Using Mass Spectrometry and 31P Nuclear Magnetic Resonance

“…Mass spectrometry (MS) has been shown to be a suitable tool to identify and verify spin adducts. these techniques were applied to the characterization of radical adducts of the nitrones benzylidene(tert-butyl)azane oxide (PBN) 15,16 , 4-{[tertbutyl(oxido)imino]-methyl} pyridine 1-oxide (PoBN), [17][18][19][20] 2,2 dimethyl-3,4-dihydropyrroline N-oxide (DMPo), [21][22][23][24][25][26][27] N-arylc,c-dimethoxycarbonylnitrones 28 and (2-methyl-1-oxido-3,4-dihydro-2H-pyrrol-2-yl) phosphonate (DEPMPo). 29 the mass spectrometric analyses were performed on the isolated radical spin adducts using high-performance liquid chromatography (HPLc) or with on-line chromatographic system such as HPLc or Gc.…”

“…[15][16][17][18][19][20][21] recently, the structural determination of free radical spin adducts from a complex mixture has been reported without any preliminary separation. [22][23][24][25][26][27][28][29] Each method presents different advantages and disadvantages. Isolation with chromatographic systems is a long procedure and does not permit the analysis of the initial paramagnetic spin adducts.…”

Structural Analysis of Amino Acids, Oxidized by Reactive Oxygen Species and an Antibody against N-Formylkynurenine

“…However, to quantify the individual oxidation products, the development of more advanced technology is awaited [25]. The data presented here suggest that the antiserum recognizes NFK on an oxidized protein as well as kynurenine-bound proteins.…”