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Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides

Nature Biotechnology volume 21, pages 566569 (2003) | Download Citation



Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system1,2 or it is first reduced in complexity by isolating sets of representative peptides3,4,5,6,7,8. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis9 and diagonal chromatography10. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides11. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation12 and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS)13,14; they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

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K.G. is a postdoctoral fellow and L.M. a research assistant of the Fund for Scientific Research–Flanders (Belgium) (F.W.O.–Vlaanderen). The project was further supported by the Concerted Research Actions (GOA) of the Flemish Community, the Inter University Attraction Poles (IUAP), and the GBOU-research initiative of the Flanders Institute of Science and Technology (IWT).

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  1. Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium.

    • Kris Gevaert
    • , Marc Goethals
    • , Lennart Martens
    • , Jozef Van Damme
    • , An Staes
    • , Grégoire R. Thomas
    •  & Joël Vandekerckhove


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The authors declare no competing financial interests.

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Correspondence to Kris Gevaert.

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