Solid-Phase Cross-Linking (SPCL): A new tool for protein structure studies

Authors

  • David Paramelle,

    1. Institut des Biomolécules Max Mousseron, CNRS, Universités Montpellier 1 et 2, Montpellier, France
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  • Christine Enjalbal,

    1. Institut des Biomolécules Max Mousseron, CNRS, Universités Montpellier 1 et 2, Montpellier, France
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  • Muriel Amblard,

    1. Institut des Biomolécules Max Mousseron, CNRS, Universités Montpellier 1 et 2, Montpellier, France
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  • Eric Forest,

    1. Proteins Mass Spectrometry Laboratory, Institut de Biologie Structurale, CEA, CNRS, UJF, Grenoble, France
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  • Michaël Heymann,

    1. Institut de Biologie et Chimie des Protéines, Laboratoire de Bioinformatique et RMN structurales, Pole BioInformatique Lyonnais, Université Lyon 1, Lyon, France
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  • Sonia Cantel,

    1. Institut des Biomolécules Max Mousseron, CNRS, Universités Montpellier 1 et 2, Montpellier, France
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  • Christophe Geourjon,

    1. Institut de Biologie et Chimie des Protéines, Laboratoire de Bioinformatique et RMN structurales, Pole BioInformatique Lyonnais, Université Lyon 1, Lyon, France
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  • Jean Martinez,

    1. Institut des Biomolécules Max Mousseron, CNRS, Universités Montpellier 1 et 2, Montpellier, France
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    • These authors have contributed equally to this study.

  • Gilles Subra

    Corresponding author
    1. Institut des Biomolécules Max Mousseron, CNRS, Universités Montpellier 1 et 2, Montpellier, France
    • Institut des Biomolécules Max Mousseron, UMR 5247 CNRS, Universités Montpellier 1 et 2, Faculté de Pharmacie, 15 avenue Charles Flahault, 34093 Montpeller Cedex 05, France Fax: +33-4-67-54-86-54
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    • These authors have contributed equally to this study.


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Abstract

A wide range of chemical reagents are available to study the protein–protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.

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