title: High resolution mass spectrometry characterization of the oxidation pattern of methionine and cysteine residues in rat liver mitochondria voltage-dependent anion selective channel 3 (VDAC3).
abstract: Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane. In comparison with the most abundant isoform VDAC1, there is little knowledge about the functional role of VDAC3. Unlikely VDAC1, cysteine residues are particularly abundant in VDAC3. Since the mitochondrial intermembrane space (IMS) has an oxidative potential we questioned whether the redox state of VDAC3 can be modified. By means of SDS-PAGE separation, tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS analysis we investigated the oxidation state of cysteine and methionine residues of rat liver VDAC3. Our results demonstrate that the mitochondrial VDAC3, in physiological state, contains methionines oxidized to methionine sulfoxide. Furthermore, cysteine residues 36, 65, and 165 are oxidized to a remarkable extend to sulfonic acid. Cysteines 2 and 8 are observed exclusively in the carboxyamidomethylated form. Cys229is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys122could not be determined because peptides containing this residue were not detected. Control experiments ruled out the possibility that over-oxidation of cysteines might be due to artefactual reasons. The peculiar behavior of Met and Cys residues of VDAC3 may be related with the accessibility of the protein to a strongly oxidizing environment and may be connected with the regulation of the activity of this trans-membrane pore protein.
authors.affiliation: Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Electronic address: rsaletti@unict.it.
authors.forename: Rosaria
authors.initials: R
authors.lastname: Saletti
authors.affiliation: Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology, University of Catania, Viale A. Doria 6, 95125 Catania, Italy; National Institute for Biomembranes and Biosystems, Section of Catania, Viale A. Doria 6, 95125 Catania, Italy. Electronic address: simonareina@yahoo.it.
authors.forename: Simona
authors.initials: S
authors.lastname: Reina
authors.affiliation: Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Viale A. Doria 6, 95125 Catania, Italy; Department of Biomedicine and Biotechnology, Section of Biology and Genetics, Viale A. Doria 6, 95125 Catania, Italy. Electronic address: marinella.pitt@virgilio.it.
authors.forename: Maria G G
authors.initials: MG
authors.lastname: Pittalà
authors.affiliation: National Institute for Biomembranes and Biosystems, Section of Catania, Viale A. Doria 6, 95125 Catania, Italy; Department of Biomedicine and Biotechnology, Section of Biology and Genetics, Viale A. Doria 6, 95125 Catania, Italy. Electronic address: belfi88@hotmail.it.
authors.forename: Ramona
authors.initials: R
authors.lastname: Belfiore
authors.affiliation: Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Electronic address: vcunsolo@unict.it.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cunsolo
authors.affiliation: Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology, University of Catania, Viale A. Doria 6, 95125 Catania, Italy; National Institute for Biomembranes and Biosystems, Section of Catania, Viale A. Doria 6, 95125 Catania, Italy. Electronic address: mess@unict.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Messina
authors.affiliation: National Institute for Biomembranes and Biosystems, Section of Catania, Viale A. Doria 6, 95125 Catania, Italy; Department of Biomedicine and Biotechnology, Section of Biology and Genetics, Viale A. Doria 6, 95125 Catania, Italy. Electronic address: vdpbiofa@unict.it.
authors.forename: Vito
authors.initials: V
authors.lastname: De Pinto
authors.affiliation: Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Electronic address: sfoti@unict.it.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Foti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008715:Methionine / Q000737:chemistry*; D008930:Mitochondria, Liver / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis; D051381:Rats; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D010455:Peptides; C494769:Vdac3 protein, rat; D050994:Voltage-Dependent Anion Channels; D008715:Methionine; D014357:Trypsin; D003545:Cysteine
keywords: Amino acid redox state; Cysteine; Mitochondrial intermembrane space; Mitochondrial outer membrane; Orbitrap tribrid mass spectrometer; Voltage dependent anion selective channel isoform 3 (VDAC3)