TY - JOUR
T1 - Thiol oxidation and di-tyrosine formation in human plasma proteins induced by inflammatory concentrations of hypochlorous acid
AU - Colombo, Graziano
AU - Clerici, Marco
AU - Altomare, Alessandra
AU - Rusconi, Francesco
AU - Giustarini, Daniela
AU - Portinaro, Nicola
AU - Garavaglia, Maria Lisa
AU - Rossi, Ranieri
AU - Dalle-Donne, Isabella
AU - Milzani, Aldo
PY - 2017/1/30
Y1 - 2017/1/30
N2 - 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. Biological significance 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.
AB - 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. Biological significance 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.
KW - Di-tyrosine
KW - Hypochlorous acid
KW - Protein aggregates
KW - Protein carbonyls
KW - Protein thiols
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U2 - 10.1016/j.jprot.2016.10.008
DO - 10.1016/j.jprot.2016.10.008
M3 - Article
AN - SCOPUS:84993985121
VL - 152
SP - 22
EP - 32
JO - Journal of Proteomics
JF - Journal of Proteomics
SN - 1874-3919
ER -