title: Chromatographic separations as a prelude to two-dimensional electrophoresis in proteomics analysis.
abstract: Current methods of proteome analysis rely almost solely on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by the excision of individual spots and protein identification using mass spectrometry (MS) and database searching. 2-D PAGE is denaturing in both dimensions and, thus, cannot indicate functional associations between individual proteins. Moreover, less abundant proteins are difficult to identify. To simplify the proteome, and explore functional associations, nondenaturing anion exchange column chromatography was used to separate a soluble protein extract from Escherichia coli. Successive fractions were then analysed using 2-D PAGE and selected spots from both the gels for the start material and the fractionated material were quantified and identified by peptide mass fingerprinting using a MALDI-TOF mass spectrometer. Enrichments of up to 13-fold were attained for individual protein spots and peptide mass fingerprints were of significantly higher quality after chromatographic separation. The marked anomalies between predicted p/and column elution position contrasted with the almost perfect correlation with migration distance on isoelectric focusing (IEF) and were explored further for basic proteins.
authors.affiliation: Department of Biomolecular Sciences, UMIST, School of Biological Sciences, University of Manchester, Manchester, UK.
authors.forename: A
authors.initials: A
authors.lastname: Butt
authors.forename: M D
authors.initials: MD
authors.lastname: Davison
authors.forename: G J
authors.initials: GJ
authors.lastname: Smith
authors.forename: J A
authors.initials: JA
authors.lastname: Young
authors.forename: S J
authors.initials: SJ
authors.lastname: Gaskell
authors.forename: S G
authors.initials: SG
authors.lastname: Oliver
authors.forename: R J
authors.initials: RJ
authors.lastname: Beynon
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification*; D016680:Genome, Bacterial; D010449:Peptide Mapping; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome