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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS

Location: Molecular Characterization of Foodborne Pathogens

Title: Comparative proteomics of E. coli O157:H7: two-dimensional gel electrophoresis vs. two-dimensional liquid chromatography separation

Authors
item Gunther, Nereus
item Pang, Hoan-Jen - USDA HUMAN NUTR. CENTER
item Nunez, Alberto
item Uhlich, Gaylen

Submitted to: Open Proteomics Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 9, 2010
Publication Date: September 10, 2010
Citation: Gunther, N.W., Pang, H., Nunez, A., Uhlich, G.A. 2010. Comparative proteomics of E. coli O157:H7: two-dimensional gel electrophoresis vs. two-dimensional liquid chromatography separation. Open Proteomics Journal. 3:26-34.

Interpretive Summary: The ability to understand how harmful bacteria react and respond to changing environments and external stresses is crucial to controlling these organisms. Bacteria respond to different environments by varying expression of their genes and therefore changing the types and amounts of proteins that they produce. Comparing and measuring the complement of proteins bacteria produce allows us to understand the mechanisms by which harmful bacteria are able to persist and cause disease; thus allowing us the opportunity to develop strategies to control or prevent their growth. This work compares and contrasts two common technologies used for the separation, comparison, and measurement of protein expression in a food-bourne pathogenic bacterium (E. coli O157:H7) under conditions pertinent to food safety. Both methods produced data that were instructive as to how this bacterium is able to persist in various challenging environments. It is the conclusion of this work that the techniques are sufficiently different in terms of the methodologies and resulting information that they should be used in conjunction with one another to accomplish a more complete understanding of situational bacterial protein expression.

Technical Abstract: The accepted method for comparing bacterial proteomes has traditionally been two-dimensional gel electrophoresis (2-D GE). However, in recent years, new procedures for protein separation have been introduced. One of these new procedures utilizes column-based liquid chromatography (2-D LC) separation. The techniques by which these two methods separate proteins differ significantly; however, it is currently unclear to what degree the sets of proteins identified by these different methods will diverge. To answer this question we compared the proteomes of Escherichia coli O157:H7 strain EDL933 against a variant containing a point mutation in the promoter of the csgD transcriptional regulator, using both 2-D GE and the column-based system. Whole protein samples were prepared from the wild type and mutant and then split in half, with one half analyzed by 2-D GE and the other with the 2-D LC. Differentially regulated proteins were observed in each system and identified by MALD/I-Tof/Tof analysis. The differences in the protein detection sensitivities of Coomassie blue-stains used in 2-D GE and UV detectors used in column-based systems resulted in different numbers of total proteins visualized in each system and therefore different numbers of matched protein pairs visualized by each system during comparative assays. Despite the differences in numbers of proteins visualized and paired in comparative assays performed using each technique the number of differentially regulated proteins from each method that could eventually be identified by MALDI analysis were very similar for both 2-D GE and 2-D LC. Nevertheless, a lack of significant redundancy between the sets of proteins identified suggests that these two methods are complimentary and not strictly corroborative.

Last Modified: 9/10/2014
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