High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

Abstract

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.

Membrane proteome of the green sulfur bacterium Chlorobium tepidum (syn. Chlorobaculum tepidum) analyzed by gel-based and gel-free methods

“…This analysis is unique because it facilitates functional analysis of membrane protein complexes on 2D gels and has been successfully applied for the soluble proteome of C. tepidum (Aivaliotis et al 2006a). BN-PAGE is an electrophoresis technique capable of separating native, catalytically active membrane protein complexes Kantzilakis et al 2007;Nijtmans et al 2002;Schaegger and von Jagow 1991).…”

The Chaperonin Containing TCP1 Complex (CCT/TRiC) Is Involved in Mediating Sperm-Oocyte Interaction

“…The isolation of such complexes is facilitated by solubilization of cellular membranes in a weak, nonionic detergent that maintains proteinprotein interactions (30). Although originally developed for the analysis of the large multienzyme complexes comprising the mitochondrial electron transport chain (31,32), BN-PAGE has since been successfully adapted for the isolation and functional characterization of protein complexes embedded within the plasma membrane of a variety of cell types (31,33,34). Utilizing this technique, we have been able to substantiate the presence of chaperone-laden protein complexes on the surface of capacitating mouse spermatozoa and demonstrate that a subset of these complexes display high affinity binding to the zona pellucida.…”

Investigation of Protein-Protein Interactions by Blue Native-PAGE & Mass Spectrometry

“…Compared to previous methods used to study PPIs, there are several advantages of BN-PAGE for studying protein-protein interactions: 1) separation of protein complexes takes place under native conditions, so even transient interactions between proteins may be identified, 2) the method may simultaneously analyze association into-or dissociation from protein complexes of particular proteins as a result of ligand stimulation (e.g. time course analysis of a particular protein complex), 3) By combining BN-PAGE with liquidchromatography-tandem mass spectrometry (LC-MS/MS), both structural and functional information may be obtained [17,[20][21][22].…”