Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns

Abstract

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.

TheEscherichia coliProteome: Past, Present, and Future Prospects

“…Thus, the cellular responses, as reflected by the proteome, can differ In one of the first studies using proteomics, comparison of 2-D gels allowed identification of a large group of E. coli heat shock proteins (166,208,210,296). In the following years, many E. coli proteomic studies revealed changes in proteome profiles in response to various stresses, such as changes in pH (24,27,274,323), cell density (70,325), and temperature (109,291); organic solvents (321); nutrient starvation (293,311); and anaerobic conditions (271) ( Table 2). These studies resulted in the identification of various E. coli stress-induced stimulons (Fig.…”

“…Among these, Adi, AstD, CadA, CysK, GabT, SpeF, and TnaA were also significantly induced by anaerobiosis. Recently, researchers have used enrichment by column chromatography on reactive dye columns for the proteomic investigation of lowabundance acid-responsive proteins in E. coli grown at either pH 7.0 or pH 5.8 (24). This work allowed identification of new pH-responsive proteins: six acid-induced proteins (EF-Ts, GdhA, PanC, ProC, TkrA, and YodA) and five acid-repressed proteins (AroG, EF-Tu, FabI, GlyA, and PurA).…”

Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance

“…Protein expression differences greater than twofold that were obtained for all six pairs of replicates were considered significant. The proteins were identified using a combination of tryptic digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry, as previously described (10).…”

Silica based click amino stationary phase for ion chromatography and hydrophilic interaction liquid chromatography

“…7 In addition, quaternary ammonium, sulfonic and zwitterionic group-functionalized polymers or silica beads were also used for analysis of ionic compounds. [8][9][10][11][12] Recently a variety of HILIC stationary phases have been developed for different purposes, such as separation of polar compounds and peptides by multi-dimensional separation techniques, 13-20 enrichment of lower abundance biological compounds by HILIC solid-phase extraction (SPE), including glycopeptides, proteins and glycoproteins, [21][22][23] as well as analysis of complicated polar samples by using HILIC-MS hyphenated technique, due to good compatibility between organic mobile phase used in HILIC with MS. 24 A common characteristic between IC and HILIC stationary phases is that both contain polar or hydrophilic groups. In IC, ion exchange occurs between charged or chargeable moieties of the stationary phase and the ionic analytes, while HILIC displayed both electrostatic interaction and partition interaction between stationary phase and analytes.…”