H-Ala-Trp-Ala-OH

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the α-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and β-enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C₁₈-phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan.

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.

We describe the characterization of degradation products responsible for color change in near UV-visible light-irradiated and heat-stressed monoclonal antibody (mAb) drug product in liquid formulation. The treated samples were characterized using reversed-phase HPLC and size-exclusion HPLC with absorption spectroscopy. Both methods showed color change was due to chromophores formed on the mAb but not associated with the formulation excipients in both light-irradiated and heat-stressed mAb samples. These chromophores were further located by a new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy. Mass spectrometry identified the major tryptophan oxidation products as kynurenine (Kyn), N-formylkynurenine (NFK), and hydroxytryptophan (OH-Trp). The absorption spectra showed that each of the tryptophan oxidation products exhibited a distinct absorption band above 280 nm shifted to the longer wavelengths in the order of OH-Trp < NFK < Kyn. The Kyn-containing peptide was detected by absorption at 420 nm. No new absorption bands were observed for either methionine or histidine oxidation products. This confirmed that tryptophan oxidation products, but not methionine and histidine oxidation products, were responsible for the color change. It is worth noting that a new oxidation product with the loss of hydrogen (2 Da mass decrease) for Trp-107 of the heavy chain was identified in the heat-stressed mAb sample. This oxidized tryptophan residue exhibited a distinct absorption band at the maximum absorbance wavelength 335 nm, which is responsible for the color change to yellow. This study showed that the new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy is useful to identify tryptophan oxidation products as chromophores responsible for color change in stressed mAb drug product.