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Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening

Authors

  • Alexander Beck,

    Corresponding author
    1. Klinisch-chemisches Zentrallabor der Universitätskliniken, Abteilung Innere Medizin IV, Universität Tübingen, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany
    2. Abteilung für Physikalische Biochemie, Physiologisch-chemisches Institut der Universität Tübingen, Hoppe-Seyler-Str. 4, D-72076 Tübingen, Germany
    • Zentrallabor/Abt. Innere Med. IV, Universitätskliniken Tübingen, Otfried-Müller-Str. 10, D-72076 Tübingen, Germany.
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  • Martin Deeg,

    1. Interdisziplinäres klinisches Forschungszentrum der Universitätskliniken, Waldhörnlestr. 22, D-72072 Tübingen, Germany
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  • Klaus Moeschel,

    1. Klinisch-chemisches Zentrallabor der Universitätskliniken, Abteilung Innere Medizin IV, Universität Tübingen, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany
    2. Abteilung für Physikalische Biochemie, Physiologisch-chemisches Institut der Universität Tübingen, Hoppe-Seyler-Str. 4, D-72076 Tübingen, Germany
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  • Eckhart K. Schmidt,

    1. Interdisziplinäres klinisches Forschungszentrum der Universitätskliniken, Waldhörnlestr. 22, D-72072 Tübingen, Germany
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  • Erwin D. Schleicher,

    1. Klinisch-chemisches Zentrallabor der Universitätskliniken, Abteilung Innere Medizin IV, Universität Tübingen, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany
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  • Wolfgang Voelter,

    1. Abteilung für Physikalische Biochemie, Physiologisch-chemisches Institut der Universität Tübingen, Hoppe-Seyler-Str. 4, D-72076 Tübingen, Germany
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  • Hans U. Häring,

    1. Klinisch-chemisches Zentrallabor der Universitätskliniken, Abteilung Innere Medizin IV, Universität Tübingen, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany
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  • Rainer Lehmann

    1. Klinisch-chemisches Zentrallabor der Universitätskliniken, Abteilung Innere Medizin IV, Universität Tübingen, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany
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Abstract

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS).1,, 2 Phosphorylation-specific marker ions (m/z 79, PO3, and m/z 97, H2PO4) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO3) and m/z 97 (H2PO4) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C2F3O fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic β-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by µLC/ESI-sCID-MS and µLC/ESI-MS analysis. Copyright © 2001 John Wiley & Sons, Ltd.

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