Structural characterization of intra- and intermolecular disulfide bonds in voltage-dependent anion channel 3 (VDAC3) protein from Rattus norvegicus by high-resolution mass spectrometry.

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They participate in the control of glycolytic metabolism through interaction with numerous enzymes and play a key role in the regulation of mitochondria-mediated apoptosis, cancer, and neurodegenerative diseases. The enzymatic digestion procedure in solution, originally developed in our laboratory, followed by high-resolution mass spectrometry coupled with UHPLC, has proven to be a powerful tool for the structural characterization of these \"difficult\" proteins. In this work, we used this procedure for the localization of intramolecular disulfide bonds in rVDAC3 and also for the 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 identifications were not supported by MS/MS spectra. The formation of intermolecular disulfide bonds helps to explain the previously observed VDAC oligomerization and demonstrates that disulfide bridges are directly involved in their homo- or hetero-oligomerization. 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