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

Rosslyn Margaret Birch, C. O'Byrne, Ian Rylance Booth, Phillip Cash

Research output: Contribution to journalArticle

54 Citations (Scopus)

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 YbdA) were observed for E coli grown at pH 5.8. Five identified proteins (AroG, Fabl, 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 Fabl 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.

Original languageEnglish
Pages (from-to)764-776
Number of pages12
JournalProteomics
Volume3
Issue number5
DOIs
Publication statusPublished - May 2003

Keywords

  • acid habitation
  • chromatographic enrichment
  • Escherichia coli
  • proteome
  • 2-dimensional gel-electrophoresis
  • immobilized PH gradients
  • low-abundance proteins
  • cibacron blue F3GA
  • procion red HE-3B
  • haemophilus-influenzae
  • mass-spectrometry
  • affinity-chromatography
  • Salmonella-Typhimmurium
  • Heparin chromatography

Cite this

Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns. / Birch, Rosslyn Margaret; O'Byrne, C.; Booth, Ian Rylance; Cash, Phillip.

In: Proteomics, Vol. 3, No. 5, 05.2003, p. 764-776.

Research output: Contribution to journalArticle

Birch, Rosslyn Margaret ; O'Byrne, C. ; Booth, Ian Rylance ; Cash, Phillip. / Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns. In: Proteomics. 2003 ; Vol. 3, No. 5. pp. 764-776.
@article{96be2dffdca24e05b2b1233e7af82dc1,
title = "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 YbdA) were observed for E coli grown at pH 5.8. Five identified proteins (AroG, Fabl, 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 Fabl 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.",
keywords = "acid habitation, chromatographic enrichment, Escherichia coli, proteome, 2-dimensional gel-electrophoresis, immobilized PH gradients, low-abundance proteins, cibacron blue F3GA, procion red HE-3B, haemophilus-influenzae, mass-spectrometry, affinity-chromatography, Salmonella-Typhimmurium, Heparin chromatography",
author = "Birch, {Rosslyn Margaret} and C. O'Byrne and Booth, {Ian Rylance} and Phillip Cash",
year = "2003",
month = "5",
doi = "10.1002/pmic.200300397",
language = "English",
volume = "3",
pages = "764--776",
journal = "Proteomics",
issn = "1615-9853",
publisher = "John Wiley & Sons, Ltd",
number = "5",

}

TY - JOUR

T1 - Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns

AU - Birch, Rosslyn Margaret

AU - O'Byrne, C.

AU - Booth, Ian Rylance

AU - Cash, Phillip

PY - 2003/5

Y1 - 2003/5

N2 - 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 YbdA) were observed for E coli grown at pH 5.8. Five identified proteins (AroG, Fabl, 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 Fabl 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.

AB - 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 YbdA) were observed for E coli grown at pH 5.8. Five identified proteins (AroG, Fabl, 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 Fabl 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.

KW - acid habitation

KW - chromatographic enrichment

KW - Escherichia coli

KW - proteome

KW - 2-dimensional gel-electrophoresis

KW - immobilized PH gradients

KW - low-abundance proteins

KW - cibacron blue F3GA

KW - procion red HE-3B

KW - haemophilus-influenzae

KW - mass-spectrometry

KW - affinity-chromatography

KW - Salmonella-Typhimmurium

KW - Heparin chromatography

U2 - 10.1002/pmic.200300397

DO - 10.1002/pmic.200300397

M3 - Article

VL - 3

SP - 764

EP - 776

JO - Proteomics

JF - Proteomics

SN - 1615-9853

IS - 5

ER -