Comparative Proteomics of E. Coli O157:H7: Two-Dimensional Gel Electrophoresis vs. Two-Dimensional Liquid Chromatography Separation

ABSTRACT

The 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 address this question we compared the proteomes of Escherichia coli O157:H7 strain EDL933 against a naturally occurring variant using both 2-D GE and 2-D LC. Whole protein samples were prepared from the wild type and variant and 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 stain used in 2-D GE and UV detectors used for 2-D LC resulted in different numbers of total proteins visualized in each system and therefore different numbers of matched protein pairs visualized during comparative assays. Despite the differences in visualization the numbers, but not the identities, of the differentially regulated proteins that could be identified by MALD/I analyses were similar for both 2-D GE and 2-D LC. However, a lack of significant redundancy between the sets of proteins identified suggests that these two methods are complimentary and not strictly corroborative.

Keywords: