A complexomic study of Escherichia coli using two‐dimensional blue native/SDS polyacrylamide gel electrophoresis

Abstract

Study of the complexome - all the protein complexes of the cell - is essential for a better understanding and more global vision of cell function. Using two-dimensional blue native/SDS-PAGE (2-D BN/SDS-PAGE) technology, the cytosolic and membrane protein complexes of Escherichia coli were separated. Then, the different partners of each protein complex were identified by LC-MS/MS. In this report, 306 protein complexes were separated and identified. Among these protein complexes, 50 heteromultimeric and 256 homomultimeric protein complexes were found. Among the 50 heteromultimeric protein complexes, 18 previously described protein complexes validate the technology. In this study, 109 new protein complexes were found, providing insight into the function of previously uncharacterized bacterial proteins.

Damage of lipopolysaccharides in outer cell membrane and production of ROS-mediated stress within bacteria makes nano zinc oxide a bactericidal agent

“…250 lL of 19 SDS-PAGE loading buffer was added to each cell pellets and vortex until cells were resuspended. Then cells were boiled 5 min in H 2 O bath, the samples were centrifuged at 300 rpm and supernatants were used for 12 % SDS-PAGE study (Lasserre et al 2006). …”

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS

“…In E. coli there was only a limited overlap of 9 integral membrane proteins between the datasets obtained after SDS-TFE-SDS-PAGE and BN-SDS-PAGE separation [5,12,13], indicating that in this organism the approaches are complementary. In yeast mitochondria, only one TFEsensitive protein not integral to the membrane could be identified using our approach.…”

“…When applied to E. coli inner membrane preparations, 23 integral membrane proteins and 16 peripheral membrane proteins were identified in off-diagonal spots, while only 14 soluble proteins were found. Comparison to the results of two BN-SDS-PAGE studies on E. coli [12,13], revealed an overlap of only 9 integral membrane proteins and 11 peripheral proteins, indicating that, particularly for integral membrane proteins, the two techniques are complementary. For two of the proteins identified in the SDS-TFE-SDS-PAGE study, the preprotein translocase SecA and the mechanosensitive channel MscS, the oligomeric state and, in the latter case, its dissociation by TFE was confirmed in one dimensional SDS-PAGE gels using immunodetection [8,14].…”

Preparation and Characterization of Bacterial Protein Complexes for Structural Analysis

“…The agreement between different methods applied to the same genome is not particularly good, although in a favorable case of 31 proteins from the H. pylori type IV secretion system (Terradot et al, 2004) co-purification experiments authenticated 76% of the interactions predicted from large-scale Y2H experiments (Rain et al, 2001). Blue native/SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) (Wittig and Schagger, 2008) has been used to identify additional protein complexes from H. pylori (Lasserre et al, 2006).…”

“…It has also been shown that proteins that participate in the same interaction share similar mRNA half-lives, a relationship not previously noted (Janga and Babu, 2009). An alternative approach, the use of blue native/SDS-PAGE, followed by the identification of the protein complex by LC-MS/MS, has been used to identify hetero-oligomeric complexes from E. coli (Lasserre et al, 2006). Other studies have focused on characterizing another, although more experimentally challenging set of protein complexes, those found in the membrane (Stenberg et al, 2005).…”