TY - JOUR

T1 - Selective and sensitive UHPLC-ESI-Orbitrap MS method to quantify protein oxidation markers

AU - Poojary, Mahesha M.

AU - Tiwari, Brijesh K.

AU - Lund, Marianne N.

N1 - Publisher Copyright: © 2021 The Authors

PY - 2021

Y1 - 2021

N2 - 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.

AB - 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.

KW - Analytical method validation

KW - Protein hydrolysis

KW - Protein nitration

KW - Protein oxidation

KW - Reactive oxygen species

KW - Tryptophan metabolism

U2 - 10.1016/j.talanta.2021.122700

DO - 10.1016/j.talanta.2021.122700

M3 - Journal article

C2 - 34364496

AN - SCOPUS:85110166339

VL - 234

JO - Talanta

JF - Talanta

SN - 0039-9140

M1 - 122700

ER -