title: Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins.
abstract: A novel gel-free proteomic technology was used to identify more than 800 proteins from 50 million Escherichia coli K12 cells in a single analysis. A peptide mixture is first obtained from a total unfractionated cell lysate, and only the methionine-containing peptides are isolated and identified by mass spectrometry and database searching. The sorting procedure is based on the concept of diagonal chromatography but adapted for highly complex mixtures. Statistical analysis predicts that we have identified more than 40% of the expressed proteome, including soluble and membrane-bound proteins. Next to highly abundant proteins, we also detected low copy number components such as the E. coli lactose operon repressor, illustrating the high dynamic range. The method is about 100 times more sensitive than two-dimensional gel-based methods and is fully automated. The strongest point, however, is the flexibility in the peptide sorting chemistry, which may target the technique toward quantitative proteomics of virtually every class of peptides containing modifiable amino acids, such as phosphopeptides, amino-terminal peptides, etc., adding a new dimension to future proteome research.
authors.affiliation: Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University and Flanders Interuniversity Institute for Biotechnology, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Damme
authors.forename: Marc
authors.initials: M
authors.lastname: Goethals
authors.forename: Grégoire R
authors.initials: GR
authors.lastname: Thomas
authors.forename: Bart
authors.initials: B
authors.lastname: Hoorelbeke
authors.forename: Hans
authors.initials: H
authors.lastname: Demol
authors.forename: Lennart
authors.initials: L
authors.lastname: Martens
authors.forename: Magda
authors.initials: M
authors.lastname: Puype
authors.forename: An
authors.initials: A
authors.lastname: Staes
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
mesh_terms: D000465:Algorithms; D002845:Chromatography / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000302:isolation & purification; D008715:Methionine / Q000032:analysis*; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis*
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D020543:Proteome; D008715:Methionine