Collision-induced dissociative chemical cross-linking reagent for protein structure characterization: applied Edman chemistry in the gas phase

Authors

  • Frank Dreiocker,

    1. Department of Chemistry, Institute of Organic Chemistry, University of Cologne, Greinstraße 4, D-50939 Cologne, Germany
    Search for more papers by this author
    • Both authors contributed equally to this work.

  • Mathias Q. Müller,

    1. Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, D-06120 Halle (Saale), Germany
    Search for more papers by this author
    • Both authors contributed equally to this work.

  • Andrea Sinz,

    Corresponding author
    1. Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, D-06120 Halle (Saale), Germany
    • Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, D-06120 Halle (Saale), Germany.
    Search for more papers by this author
  • Mathias Schäfer

    Corresponding author
    1. Department of Chemistry, Institute of Organic Chemistry, University of Cologne, Greinstraße 4, D-50939 Cologne, Germany
    • Department of Chemistry, Institute of Organic Chemistry, University of Cologne, Greinstraße 4, D-50939 Cologne, Germany.
    Search for more papers by this author

Abstract

Chemical cross-linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross-linked products presents an alternative approach to assess low-resolution protein structures and to gain insight into protein interfaces. In this contribution, we report the design of an innovative cross-linker based on Edman degradation chemistry, which leads to the formation of indicative mass shifted fragment ions and constant neutral losses (CNLs) in electrospray ionization (ESI)-tandem-mass spectrometry (MS/MS) product ion mass spectra, allowing an unambiguous identification of cross-linked peptides. Moreover, the characteristic neutral loss reactions facilitate automated analysis by multiple reaction monitoring suited for high throughput studies with good sensitivity and selectivity. The functioning of the novel cross-linker relies on the presence of a highly nucleophilic sulfur in a thiourea moiety, safeguarding for effective intramolecular attack leading to predictive and preferred cleavage of a glycyl-prolyl amide bond. Our innovative analytical concept and the versatile applicability of the collision-induced dissociative chemical cross-linking reagent are exemplified for substance P, luteinizing hormone releasing hormone LHRH and lysozyme. The novel cross-linker is expected to have a broad range of applications for probing protein tertiary structures and for investigating protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.

Ancillary