Abstract
VDACs (voltage dependent anion channels) are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through mitochondrial membranes. The functional role of VDAC has been investigated in many studies, and different functions of VDAC have been shown. Posttranslational modifications of VDAC are significant for its regulation. The aim of our research was to develop approach for characterization of primary structure and posttranslational modifications of VDACs and other membrane proteins. Mouse brain membranes were isolated from mouse brains by differential centrifugation. Primary structure of mitochondrial isoform VDAC1 from mouse brain membranes has been identified almost completely (95 %, 258 of 283 amino acids) by combination of SDS-PAGE and LTQ-FTMS mapping of peptide mixtures after proteolytic degradation with trypsin. Sequence of each found peptide of VDAC has been analysed and confirmed according to accurate mass, isotopic distribution and MS/MS tandem analysis. Posttranslational modifications of VDAC’speptides have been shown. High sequence coverage of VDAC has been obtained, including 11 transmembranes domains. Extensive sequence coverage has been also detected for some other proteins at 30-34 kDa. A repressor of estrogen receptor activity has been identified with 76 % coverage, malate dehydrogenase with 55 % sequence coverage, syntaxin 1A and syntaxin 1B2 have been sequenced with 60 % and 65 % coverage, respectively. These results demonstrated that mass spectrometric mapping is reliable and sensitive approach for characterization of primary structure membrane proteins and identification of their posttranslational modifications.
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