Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns.
The reliable identification and analysis of the low abundance proteins expressed by a cell remains a key challenge in the study of cellular proteomes. The analysis of low abundance proteins is a particular problem when using two-dimensional gel electrophoresis (2-DE) to resolve the cellular proteins since the technology is unable to display the wide dynamic range of protein levels typically synthesized by cells. We have investigated the use of reactive dye compounds for the enrichment of low abundance cellular proteins prior to analysis by 2-DE. The capacity of reactive dye compounds to bind specific protein species was used as the basis for a general chromatographic tool for protein enrichment. Six reactive dye compounds were investigated in detail for the analysis of Escherichia coli proteins. Whole bacterial cell lysates were passed down columns prepared with the reactive dye compounds. The bound proteins were eluted with 1.5 M NaCl and analyzed by 2-DE. Distinctive protein profiles were observed for the bound proteins recovered from the different reactive dye compounds. Selected proteins enriched by these methods were identified by peptide mass mapping. The enrichment procedure developed using reactive dye compounds were used to investigate acid-induced changes in the proteome of E. coli grown at either pH 7.0 or pH 5.8. Increased levels of expression were observed for a number of proteins (for example, GdhA, PanC, ProC, TkrA, EF-TS and YodA) were observed for E. coli grown at pH 5.8. Five identified proteins (AroG, FabI, GlyA, PurA and EF-Tu) showed reduced levels of synthesis for bacteria grown at pH 5.8 compared to pH 7.0. In the case of PanC and FabI the altered expression profiles were only reliably demonstrated using the enrichment protocols. One theme emerging from these data was that the expression of proteins concerned with one-carbon metabolism was perturbed at pH 5.8, which may point to a previously unrecognized affect of low pH stress on the physiology of E. coli cells. We conclude that the prefractionation of cell lysates on reactive dye columns will serve as a valuable generic tool for the analysis of low abundance proteins expressed by both prokaryotic and eukaryotic cells.
Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen, Scotland.
Rosslyn M
RM
Birch
Conor
C
O'Byrne
Ian R
IR
Booth
Phillip
P
Cash
D004396:Coloring Agents; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D010449:Peptide Mapping; D040901:Proteomics / Q000379:methods*
D004396:Coloring Agents; D029968:Escherichia coli Proteins