Reducing the complexity of the Escerichia coli proteome by chromatography on reactive dye columns

Phillip Cash, Ian Rylance Booth

Research output: Contribution to journalArticle

Abstract

High-resolution 2-dimensional gel electrophoresis (2DGE) is a key technology in the analysis of cellular proteomes particularly in the field of microbiology. However, the restricted resolution of 2DGE and the limited dynamic range of established staining methods limit its usefulness for characterising low abundance proteins. Consequently, methods have been developed to either enrich for low abundance proteins directly or to deplete the highly abundant proteins present in complex samples. We present a protocol for affinity chromatography on reactive dye resins for the analysis of the Escherichia coli proteome. Using a range of commercially available reactive dye resins in a traditional chromatography system we were able to enrich low abundance proteins to levels suitable for their reliable detection and, most importantly, their identification using standard peptide mass mapping and MALDI-TOF MS methods. Under the chromatography conditions employed up to 4.42% of the proteins present in the total nonfractionated E. coli cell lysates bound to the reactive dye column and were subsequently eluted by 1.5 M NaCl. Of the bound proteins approximately 50% were considered to be enriched compared to the nonfractionated cell lysate. The ability to detect low abundance proteins was due to a combination of the specific enrichment of the proteins themselves as well as the depletion of highly abundant cellular proteins, which otherwise obscured the low abundance proteins. There was evidence of some selectivity between the different reactive dye resins for particular proteins. However, the selection of suitable dye resins to selectively enrich for particular classes of proteins remains largely empirical at this time.
Original languageEnglish
Pages (from-to)167-185
Number of pages18
JournalMethods in Molecular Biology
Volume424
DOIs
Publication statusPublished - 25 Jan 2008

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Proteome
Chromatography
Coloring Agents
Proteins
Electrophoresis
Gels
Escherichia coli
Peptide Mapping
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Microbiology
Affinity Chromatography
Staining and Labeling
Technology

Keywords

  • Chromatography
  • protein enrichment
  • proteome
  • reactive dye columns

Cite this

Reducing the complexity of the Escerichia coli proteome by chromatography on reactive dye columns. / Cash, Phillip; Booth, Ian Rylance.

In: Methods in Molecular Biology, Vol. 424, 25.01.2008, p. 167-185.

Research output: Contribution to journalArticle

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