Membrane proteome analysis of the green‐sulfur bacterium Chlorobium tepidum

Abstract

An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including membrane proteins, which are rarely detectable on two-dimensional electrophoresis gels. In this study, different methods were employed for the enrichment of membrane proteins from Chlorobium tepidum prior to analysis with two-dimensional electrophoresis (2-DE). Isolated membranes were solubilized with Triton X-100 and from the supernatant we identified 58 unique proteins. The use of ionic sodium dodecyl sulfate (SDS) for protein solubilization, combined with acetone precipitation, resulted in an improved 2-DE pattern and the total number of the identified proteins was increased to 117. The use of acetone for protein precipitation improved the results by extracting compounds potentially deleterious to the resolution of 2-DE. However, the additional proteins detected by the use of SDS are in the majority more difficult to solubilize than less hydrophobic proteins. Further our attempts for selective extraction of the outer membrane proteins using the acid glycine method allowed the identification of 37 proteins of which 14 were predicted to have a signal sequence indicating their localization in the periplasmic space or in the outer membrane.

Carbon Source-dependent Assembly of the Snf1p Kinase Complex inCandidaalbicans

“…Deisotoped peaks were labeled by the software, and the 100 most intense peaks were used for data base searching. Autolytic tryptic peptides or peptides resulting from the identified protein were used for internal calibration (45).…”

“LANESPECTOR”, a tool for membrane proteome profiling based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis/liquid chromatography - tandem mass spectrometry analysis: Application toListeria monocytogenes membrane proteins

“…In the present study, Lmo0927 was identified by one peptide which belongs to the hydrophobic part of the protein (intersection region between TMD 3 and 4). This peptide was detected in section 11 (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) while the calculated mass of the total protein is 74.7 kDa.…”

“…However, and contrary to the robustness of expression analyses, classical 2-D PAGE still fails to surmount fundamental limitations related to the biochemical properties of the investigated proteins. Hydrophobic proteins as well as those with extreme pI or molecular weight or low-abundant proteins are generally not accessible by 2-D PAGE [9][10][11][12], although noteworthy improvements for membrane proteomics have been established by different groups [13][14][15]. While the restriction with regard to the characterization of low-abundant proteins is usually compensated for by particular biochemical procedures for enrichment of the desired subcellular fraction [16], non-gel based technologies -mainly LC-MS/MS -are generally accepted without reservations even with regard to the other limitations mentioned above [17,18].…”

Enabling Coupled Quantitative Genomics and Proteomics Analyses from Rat Spinal Cord Samples

“…Indeed it is generally recognized that specific protein losses occur with any kind of organic-based protein denaturation protocol, immediately calling into question the suitability of these extracts for truly quantitative, global proteomics analyses. Nonetheless many groups continue to routinely extract protein from biological samples using somewhat comparable organic precipitation protocols, and some select advantages to this practice are suggested (16,17).…”