title: Thiol oxidation and di-tyrosine formation in human plasma proteins induced by inflammatory concentrations of hypochlorous acid.
abstract: In this study, we assessed the oxidative damage occurring in plasma proteins when human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). We used specific thiol labelling and Western blot analyses to determine protein thiol oxidation, as well as analytical gel filtration HPLC coupled to fluorescence detection to explore formation of high molecular weight (HMW) protein aggregates. Thiol-containing proteins oxidized by HOCl were identified by redox proteomics. Mass spectrometry (MS) analysis was performed to elucidate the protein composition of HMW aggregates. α1-antitrypsin, transthyretin, and haptoglobin showed thiol oxidation at HOCl concentrations higher than those causing complete oxidation of albumin. At the highest HOCl concentrations, formation of carbonylated and di-tyrosine cross-linked HMW protein aggregates also occurred. MS analysis identified fibrinogen, complement C3 and apolipoprotein A-I as components of HMW protein aggregates. These results could be relevant for human diseases characterized by inflammatory conditions in which myeloperoxidase and HOCl are involved.
  
  
  
  In this study we evaluated the oxidative damage occurring on plasma proteins when reconstituted human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). Pathophysiological concentrations of HOCl are able to induce different modifications on plasma proteins such as carbonylation, sulfhydryl oxidation and formation of high molecular weight (HMW) protein aggregates characterized by di-tyrosine fluorescence. There are two relevant aspects emerging from this paper. The first one consists on identifying low abundant proteins undergoing sulfhydryl oxidation by biotin-maleimide derivatization followed by MALDI-TOF mass spectrometry. This approach suggests three low-abundant proteins undergoing HOCl-induced oxidation: transthyretin, α1-antitrypsin, and haptoglobin. In addition, we analysed HMW protein aggregates forming after HOCl exposure. These aggregates are characterized by carbonylation, intra- and/or intermolecular di-tyrosine bridges. After their isolation from SDS-PAGE gel electrophoresis, using electrospray tandem mass spectrometry coupled to reversed-phase nanoscale capillary liquid chromatography, we identified some protein constituents of these HMW aggregates such as α, β, γ fibrinogen chains, apolipoprotein A-I and complement C3. In particular, our work highlights how fibrinogen is an important constituent of HOCl-induced HMW protein aggregates validating the mass spectrometry result with additional experiments. Further investigations are required in order to evaluate the possibility to use carbonylated and di-Tyr cross-linked HMW protein aggregates as (early) biomarkers for disease progression in inflammatory conditions in which myeloperoxidase and HOCl are involved.
authors: Department of Biosciences, Università degli Studi di Milano, Milan, Italy. Electronic address: graziano.colombo@unimi.it.
authors: Graziano
authors: G
authors: Colombo
authors: Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
authors: Marco
authors: M
authors: Clerici
authors: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.
authors: Alessandra
authors: A
authors: Altomare
authors: Laboratory of Neuroepigenetics, Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Centro di Eccellenza per le Malattie Neurodegenerative (CEND), Università degli Studi di Milano, Milan, Italy.
authors: Francesco
authors: F
authors: Rusconi
authors: Department of Evolutionary Biology, University of Siena, Siena, Italy.
authors: Daniela
authors: D
authors: Giustarini
authors: Department of Medical Biotechnology and Translational Medicine, Clinica Ortopedica e Traumatologica, Università degli Studi di Milano, Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors: Nicola
authors: N
authors: Portinaro
authors: Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
authors: Maria Lisa
authors: ML
authors: Garavaglia
authors: Department of Evolutionary Biology, University of Siena, Siena, Italy.
authors: Ranieri
authors: R
authors: Rossi
authors: Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
authors: Isabella
authors: I
authors: Dalle-Donne
authors: Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
authors: Aldo
authors: A
authors: Milzani
mesh_terms: D016632:Apolipoprotein A-I / Q000378:metabolism; D015415:Biomarkers / Q000097:blood; D001798:Blood Proteins / Q000378:metabolism*; D001800:Blood Specimen Collection / Q000379:methods; D003176:Complement C3 / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology*; D007249:Inflammation / Q000139:chemically induced*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D009195:Peroxidase / Q000378:metabolism; D066329:Protein Aggregates / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D015415:Biomarkers; D001798:Blood Proteins; D003176:Complement C3; D066329:Protein Aggregates; D013438:Sulfhydryl Compounds; D014443:Tyrosine; D006997:Hypochlorous Acid; D005340:Fibrinogen; D009195:Peroxidase
keywords: Di-tyrosine; Hypochlorous acid; Protein aggregates; Protein carbonyls; Protein thiols