Selective and sensitive UHPLC-ESI-Orbitrap MS method to quantify protein oxidation markers

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A targeted UHPLC-MS/MS isotopic dilution method has been developed for the simultaneous quantification of 18 different free and protein-bound aromatic amino acid oxidation products in food and biological matrices. All analytes, including critical isomeric pairs of Tyr, o-Tyr, m-Tyr, and dioxyindolylalanine diastereomers were chromatographically resolved to obtain high selectivity, without the need for derivatizing or ion pairing agents. The results of method validation showed adequate retention time reproducibility [0.1–0.6% coefficient of variation (CV) for over 224 injections], accuracy (within ±1–20% of the nominal concentration), and precision (1–17% CV) for all target analytes. The lower limit of quantification was calculated in different matrices using both the Hubaux-Vos approach and accuracy and precision data showing values in the range of 0.2–15 ng/mL. Use of stable isotope-labelled internal standards compensated errors due to matrix effects and artefactual degradation of analytes. Both acid and enzymatic hydrolyses were tested to obtain the best possible results for the quantification of protein oxidation products, demonstrating the stability of target analytes under hydrolytic conditions. The method was successfully applied to quantify target analytes in serum, tissue, milk, infant formula, pork liver pâté, chicken meat and fish. The method was also applied to assess the role of Fenton's reagent in oxidizing Trp, Phe and Tyr residues in different proteins, with results showing o-Tyr, dioxyindolylalanine diastereomers, kynurenine, dityrosine being the main oxidation products. The Fenton chemistry favored the formation of o-Tyr over m-Tyr from Phe with 2–36 folds higher yields. 3-Nitrotyrosine, a marker of protein nitration, was also detected in samples treated with Fenton's reagent.

Original languageEnglish
Article number122700
Number of pages16
Publication statusPublished - 2021

Bibliographical note

Publisher Copyright:
© 2021 The Authors

    Research areas

  • Analytical method validation, Protein hydrolysis, Protein nitration, Protein oxidation, Reactive oxygen species, Tryptophan metabolism

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