Mass spectrometric mapping of ion channel proteins (porins) and identification of their supramolecular membrane assembly.
Mass spectrometric peptide mapping, particularly by matrix-assisted laser 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 separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion 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 model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions.
Bühler
S
S
Faculty of Chemistry, University of Konstanz, Germany.
Michels
J
J
Wendt
S
S
Rück
A
A
Brdiczka
D
D
Welte
W
W
Przybylski
M
M
D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D010455:Peptides; D018272:Porins