An alternative strategy for the membrane proteome analysis of the green sulfur bacterium Chlorobium tepidum using blue native PAGE and 2-D PAGE on purified membranes.
To avoid the specific problems concerning intrinsic membrane proteins in proteome analysis, an alternative strategy is described that is complementary to previous investigations using 2-D polyacrylamide gel electrophoresis (PAGE) techniques. The strategy involves (a) obtaining purified preparations of the membranes from Chlorobium tepidum by washing with 2 M NaBr, which removed membrane-associated soluble proteins and membrane-associated organelles; (b) separation of membrane protein complexes using 1-D Blue-native polyacrylamide gel electrophoresis (BN-PAGE) after solubilization with n-dodecyl-beta-d-maltoside (DDM); (c) combination of the BN with Tricine-SDS-PAGE; (d) high-throughput mass spectrometric analysis after gel band excision, in-gel digestion, and MALDI target spotting; and (e) protein identification from mixtures of tryptic peptides by peptide mass fingerprinting. Using this approach, we identified 143 different proteins, 70 of which have not been previously reported using 2-D PAGE techniques. Membrane proteins with up to 14 transmembrane helices were found, and this procedure proved to be efficient with proteins within a wide pI range (4.4-11.6). About 54% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, and protein translocation, while for the others, a function is not yet known, indicating the potential of the method for the elucidation of the membrane proteomes in general.
Aivaliotis
Michalis
M
Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Heraklion, Greece.
Karas
Michael
M
Tsiotis
Georgios
G
D001426:Bacterial Proteins / Q000032:analysis*; D002462:Cell Membrane; D041883:Chlorobium / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D020543:Proteome