MASS-SPECTROMETRIC MAPPING OF ION-CHANNEL PROTEINS (PORINS) AND IDENTIFICATION OF THEIR SUPRAMOLECULAR MEMBRANE ASSEMBLY

Mass spectrometric peptide mapping, particularly by matrix-assisted la ser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins . In combination with in situ proteolytic digestion of proteins separa ted by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping per mits identification of proteins from complex mixtures such as cell lys ates, In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial m embranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific d iffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a mod el system in this study. R.c.-porin was characterized by MALDI-MS pept ide mapping in solution, and by direct in situ-gel digestion of the tr imer. Furthermore, in this study we demonstrate the direct identificat ion of the noncovalent complex between a mitochondrial porin and the a denine nucleotide translocator from rat liver, by MALDI-MS determinati on of the specific peptides due to both protein sequences in the SDS-P AGE gel band. The combination of native gel electrophoresis and mass s pectrometric peptide mapping of the specific gel bands should be devel oped as a powerful tool for the molecular identification of protein in teractions. Proteins Suppl. 2:63-73, 1998. (C) 1998 Wiley-Liss, Inc.