Mass spectrometric characterization of peptides containing different oxidized tryptophan residues

Abstract

The term reactive oxygen species refers to small molecules that can oxidize, for example, nearby proteins, especially cysteine, methionine, tryptophan, and tyrosine residues. Tryptophan oxidation is always irreversible in the cell and can yield several oxidation products, such as 5-hydroxy-tryptophan (5-HTP), oxindolylalanine (Oia), kynurenine (Kyn), and N-formyl-kynurenine (NFK). Because of the severe effects that oxidized tryptophan residues can have on proteins, there is a great need to develop generally applicable and highly sensitive techniques to identify the oxidized residue and the oxidation product. Here, the fragmentation behavior of synthetic peptides corresponding to sequences recently identified in three skeletal muscle proteins as containing oxidized tryptophan residues were studied using postsource decay and collision-induced dissociation (CID) in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry (MS) and CID in an electrospray ionization (ESI) double quadrupole TOF-MS. For each sequence, a panel of five different peptides containing Trp, 5-HTP, Kyn, NFK, or Oia residue was studied. It was always possible to identify the modified positions by the y-series and also to distinguish the different oxidation products by characteristic fragment ions in the lower mass range by tandem MS. NFK- and Kyn-containing peptides displayed an intense signal at m/z 174.1, which could be useful in identifying accordingly modified peptides by a sensitive precursor ion scan. Most importantly, it was always possible to distinguish isomeric 5-HTP and Oia residues. In ESI- and MALDI-MS/MS, this was achieved by the signal intensity ratios of two signals obtained at m/z 130.1 and 146.1. In addition, high collision energy CID in the MALDI-TOF/TOF-MS also permitted the identification of these two isomeric residues by their v- and w-ions, respectively.

Myeloperoxidase-oxidized high density lipoprotein impairs atherosclerotic plaque stability by inhibiting smooth muscle cell migration

“…ApoA1 containing a 2-OH-W72 group is abundant in atherosclerosis-laden arteries and could predict CHD risk [9]. Here we found that MPO-catalyzed nitration and chlorination also results in the oxidation of W72 to form the singly oxidized product, W + O (16 Da), doubly oxidized product, W + 2O (32 Da), and the oxidation of W to kynurenine [30] (W + 4 Da) (Fig. 1).…”

Lipids and proteins—major targets of oxidative modifications in abiotic stressed plants

“…A number of state-of-the-art techniques, such as the reverse-phase high-performance liquid chromatography (RP-HPLC) combined with UV and fluorescence detection (Simat and Steinhart 1998;Simat et al 1994) or mass spectrometry (MS) ( Van de Weert et al 1998;Dominques et al 2003;Szuchman-Sapir et al 2008;Mouls et al 2009), are available for efficient analysis of tryptophan oxidation products. In fact, oxidized tryptophan residues, such as 5-HTP, Oia, NFK, and Kyn, have been reported to exhibit different fragmentation characteristics in both MALDIand ESI-tandem MS that in turn help in their identification by specific marker ions, in addition to their increment masses within a given ion series (Todorovski et al 2011). Todorovski et al (2011 were able to distinguish the isomeric 5-HTP-and Oiacontaining peptides.…”

“…Tyrosine and tryptophan are among amino acids that are most prone to ROS-mediated irreversible oxidation (Berlett and Stadtman 1997;Davies 2003;Stadtman and Levine 2003;Todorovski et al 2011). Indole ring is the normal target of the ROS attack on tryptophan, where 5-hydroxy-tryptophan (5-HTP), oxindolylalanine (Oia), dioxindolylalanine, N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine, and hydroxy-N-formyl-kynurenine are produced as the tryptophanoxidation products (Simat and Steinhart 1998;Garrison 1987).…”

Cardiovascular Redox and Ox Stress Proteomics

“…Several of these modified proteins include actin, troponin, H-FABP, and complex V. However, due to the multiple isomeric oxidative degradation products (e.g., serotonin or 5-hydroxytryptamine and oxyindolylalanine) that occur, it has been difficult to distinguish these in a complex protein mixture. Hoffman and colleagues (280) have shown that by using high-energy ESI-CID with isolated peptides, the differences in masses within a given ion series and specific marker ions could differentiate the isomeric forms of 5-HT and oxindolylalanine. This suggests that in complex protein mixtures, the possibility of identifying these isomeric forms is feasible.…”