title: Protein-bound tyrosine oxidation, nitration and chlorination by-products assessed by ultraperformance liquid chromatography coupled to tandem mass spectrometry.
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.
  
  
  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 (3NO2-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 3NO2-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.
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Torres-Cuevas
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain.
authors.forename: Julia
authors.initials: J
authors.lastname: Kuligowski
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Cárcel
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain.
authors.forename: Consuelo
authors.initials: C
authors.lastname: Cháfer-Pericás
authors.affiliation: Department of Physiology, University of Valencia, Vicent Andrés Estellés s/n, 46100, Burjassot, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Asensi
authors.affiliation: Department of Pediatric Research, Institute for Surgical Research, Oslo University Hospital - Rikshospitalet, Oslo, Norway.
authors.forename: Rønnaug
authors.initials: R
authors.lastname: Solberg
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain; Division of Neonatology, University & Polytechnic Hospital La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Cubells
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain; Division of Neonatology, University & Polytechnic Hospital La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Nuñez
authors.affiliation: Department of Pediatric Research, Institute for Surgical Research, Oslo University Hospital - Rikshospitalet, Oslo, Norway.
authors.forename: Ola Didrik
authors.initials: OD
authors.lastname: Saugstad
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain; Division of Neonatology, University & Polytechnic Hospital La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain; National Coordinator of the Spanish Maternal and Child Health and Developmental Network, (Retic Red SAMID RD0012/0026), Spain.
authors.forename: Máximo
authors.initials: M
authors.lastname: Vento
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain. Electronic address: justo.escobar@uv.es.
authors.forename: Javier
authors.initials: J
authors.lastname: Escobar
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002853:Chromatography, Liquid / Q000379:methods*; D054879:Halogenation; D009566:Nitrates / Q000737:chemistry*; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D009566:Nitrates; D011506:Proteins; D014443:Tyrosine
keywords: Biomarkers; Liquid chromatography; Mass spectrometry; Phenylalanine; Plasma; Protein nitration; Protein oxidation; Tissue; Tyrosine