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Chemical cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS

Authors

  • Alexandre F. Gomes,

    1. Institute of Chemistry, University of Campinas—UNICAMP and Instituto Nacional de Ciencia e Tecnologia de Bioanalitica, CP 6154, 13083-970, Campinas, Sao Paulo, Brazil
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  • Fabio C. Gozzo

    Corresponding author
    1. Institute of Chemistry, University of Campinas—UNICAMP and Instituto Nacional de Ciencia e Tecnologia de Bioanalitica, CP 6154, 13083-970, Campinas, Sao Paulo, Brazil
    • Departamento de Quimica Organica, Instituto de Quimica, Universidade Estadual de Campinas, Campinas, Sao Paulo, Brazil.
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Abstract

Crystallography and nuclear magnetic resonance are well-established methods to study protein tertiary structure and interactions. Despite their usefulness, such methods are not applicable to many protein systems. Chemical cross-linking of proteins coupled with mass spectrometry allows low-resolution characterization of proteins and protein complexes based on measuring distance constraints from cross-links. In this work, we have investigated cross-linking by means of a heterobifunctional cross-linker containing a traditional N-hydroxysuccinimide (NHS) ester and a UV photoactivatable diazirine group. Activation of the diazirine group yields a highly reactive carbene species, with potential to increase the number of cross-links compared with homobifunctional, NHS-based cross-linkers. Cross-linking reactions were performed on model systems such as synthetic peptides and equine myoglobin. After reduction of the disulfide bond, the formation of intra- and intermolecular cross-links was identified and the peptides modified with both NHS and diazirine moieties characterized. Fragmentation of these modified peptides reveals the presence of a marker ion for intramolecular cross-links, which facilitates identification. Copyright © 2010 John Wiley & Sons, Ltd.

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