Does chemical cross-linking with NHS esters reflect the chemical equilibrium of protein-protein noncovalent interactions in solution?

  • Stefanie Mädler
  • Markus Seitz
  • John Robinson
  • Renato Zenobi
Article

Abstract

Chemical cross-linking in combination with mass spectrometry has emerged as a powerful tool to study noncovalent protein complexes. Nevertheless, there are still many questions to answer. Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific cross-linking possible? To answer these questions, we performed systematic cross-linking studies with two complexes, using the N-hydroxysuccinimidyl ester disuccinimidyl suberate (DSS): (1) NCoA-1 and mutants of the interacting peptide STAT6Y, covering a KD range of 30 nM to >25 μM, and (2) α-thrombin and basic pancreatic trypsin inhibitor (BPTI), a system that shows a buffer-dependent KD value between 100 and 320 μM. Samples were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For NCoA-1· STAT6Y, a good correlation between the amount of cross-linked species and the calculated fraction of complex present in solution was observed. Thus, chemical cross-linking in combination with MALDI-MS can be used to rank binding affinities. For the mid-affinity range up to about KD ≈ 25 μM, experiments with a nonbinding peptide and studies of the concentration dependence showed that only specific complexes undergo cross-linking with DSS. To study in which affinity range specific cross-linking can be applied, the weak α-thrombin · BPTI complex was investigated. We found that the detected complex is a nonspecifically cross-linked species. Consequently, based on the experimental approach used in this study, chemical cross-linking is not suitable for studying low-affinity complexes with KD ≫ 25 μM.

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Copyright information

© American Society for Mass Spectrometry 2010

Authors and Affiliations

  • Stefanie Mädler
    • 1
  • Markus Seitz
    • 2
  • John Robinson
    • 2
  • Renato Zenobi
    • 1
  1. 1.Department of Chemistry and Applied BiosciencesZurichSwitzerland
  2. 2.Institute of Organic ChemistryUniversity of ZurichZurichSwitzerland

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