Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis - Identification of more than 800 Escherichia coli proteins

doi:10.1074/mcp.M200061-MCP200

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Submitted on September 20, 2002
Revised on October 24, 2002
Accepted on October 24, 2002

Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis - Identification of more than 800 Escherichia coli proteins

Kris Gevaert, Jozef Van Damme, Marc Goethals, Grégoire R. Thomas, Bart Hoorelbeke, Hans Demol, Lennart Martens, Magda Puype, An Staes, and Joël Vandekerckhove

Medical Protein Research, Ghent University, Gent B-9000

Corresponding Author: joel.vandekerckhove{at}rug.ac.be

A novel gel-free proteomics 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 un-fractionated 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 Escherichia coli lactose operon repressor, illustrating the high dynamic range. The method is about one hundred 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 towards 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.

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