Structural characterization of intra- and intermolecular disulfide bonds in Voltage-Dependent Anion Channel 3 (VDAC3) protein from Rattus norvegicus by high-resolution mass spectrometry

ABSTRACT: Voltage-Dependent Anion Channels, the most abundant proteins of the mitochondrial outer membrane, are responsible for exchange of ions and metabolites between cytosol and mitochondria. They participate in the control of glycolytic metabolism through interaction with numerous enzymes and play a key role in regulation of mitochondria-mediated apoptosis, cancer and neurodegenerative diseases. The structural characterization of VDACs presents difficulties because there is no established protocol for the isolation of a single VDAC isoform and/or separation from other membrane proteins, so they can only be studied as component of a mixture. Recently, we have showed that High-Resolution Mass Spectrometry coupled with UHPLC, represents a powerful tool for their structural characterization. Using this technique we discovered that cysteines in rat and human VDACs show a preferred oxidation state, ranging from reduced to trioxidized form, that is remarkably conserved between rat and human VDACs. More recently, characterization of intramolecular disulfide bonds in rVDAC2 was obtained. The successful characterization of disulfide bonds in rVDAC2, prompted us to use the same procedure for the investigation of intramolecular disulfide bonds in rVDAC3 and also to attempt a possible characterization of intermolecular disulfide bonds formed by this protein with other VDAC isoforms. As a result, three intramolecular and seven intermolecular disulfide bonds between rVDAC3 with rVDAC1 and rVDAC2 were uniquely characterized. Furthermore, evidence was obtained for the existence of two additional intramolecular disulfide bonds between Cys2/Cys8 with Cys36 and Cys122, although these identification were not supported by MS/MS spectra.

INSTRUMENT(S):

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Liver

SUBMITTER: Rosaria Saletti  

LAB HEAD: Rosaria Saletti

PROVIDER: PXD064110 | Pride | 2025-09-08

REPOSITORIES: Pride