Protein-bound tyrosine oxidation, nitration and chlorination by-products assessed by ultraperformance liquid chromatography coupled to tandem mass spectrometry

Autores de IIS La Fe

• María Isabel Torres Cuevas

  Autor

• Julia Kuligowski

  Autor

• 

  María Consuelo Cháfer Pericás

  Autor

• Miguel Angel Asensi Miralles

  Autor

• Elena Cubells Garcia

  Autor

• Antonio Guillermo Núñez Ramiro

  Autor

• Máximo Vento Torres

  Autor

• Justo Javier Escobar Cubiella

  Autor

Participantes ajenos a IIS La Fe

• Cárcel M
• Solberg R
• Saugstad OD

Grupos

• Perinatología

Abstract

Background: Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions. Objective: To develop, validate and apply a rapid method for the quantification of known hallmarks of tyrosine oxidation, nitration and chlorination in plasma and tissue proteins providing a snapshot of the oxidative stress and inflammatory status of the organism and of target organs respectively. Material and Methods: The extraction and clean up procedure entailed protein precipitation, followed by protein re-suspension and enzymatic digestion with pronase. An Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) method was developed to quantify protein released ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-nitrotyrosine (3NO(2)-Tyr) and 3-chlorotyrosine (3Cl-Tyr) as well as native phenylalanine (Phe) and tyrosine (p-Tyr) in plasma and tissue from a validated hypoxic newborn piglet experimental model. Results: In plasma there was a significant increase in the 3NO(2)-Tyr/p-Tyr ratio. On the other hand m-Tyr/Phe and 3Cl-Tyr/p-Tyr ratios were significantly increased in liver of hypoxic compared with normoxic animals. Although no significant differences were found in brain tissue, a clear tendency to increased ratios was observed under hypoxic conditions. Conclusions: UPLC-MS/MS has proven suitable for the analysis of plasma and tissue samples from newborn piglets. The analysis of biomarkers of protein oxidation, nitration and chlorination will be applied in future studies aiming to provide a deeper insight into the mechanisms of oxidation-derived protein modification caused during neonatal asphyxia and resuscitation. (C) 2016 Elsevier B.V. All rights reserved.

Keywords

• Protein oxidation; Protein nitration; Phenylalanine; Tyrosine; Liquid chromatography; Mass spectrometry; Biomarkers; Plasma; Tissue