Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC)

Authors

  • An Staes,

    1. Department of Medical Protein Research, VIB, Ghent, Belgium
    2. Department of Biochemistry, Ghent University, Ghent, Belgium
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    • Both these authors contributed equally.

  • Petra Van Damme,

    1. Department of Medical Protein Research, VIB, Ghent, Belgium
    2. Department of Biochemistry, Ghent University, Ghent, Belgium
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    • Both these authors contributed equally.

  • Kenny Helsens,

    1. Department of Medical Protein Research, VIB, Ghent, Belgium
    2. Department of Biochemistry, Ghent University, Ghent, Belgium
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  • Hans Demol,

    1. Department of Medical Protein Research, VIB, Ghent, Belgium
    2. Department of Biochemistry, Ghent University, Ghent, Belgium
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  • Joël Vandekerckhove,

    1. Department of Medical Protein Research, VIB, Ghent, Belgium
    2. Department of Biochemistry, Ghent University, Ghent, Belgium
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  • Kris Gevaert Professor

    Corresponding author
    1. Department of Medical Protein Research, VIB, Ghent, Belgium
    2. Department of Biochemistry, Ghent University, Ghent, Belgium
    • Department of Medical Protein Research and Biochemistry, VIB and Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium Fax: +32-92649496
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Abstract

We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for α-amino-blocked peptides, including α-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of α-amino-blocked and α-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting α-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are α-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

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