Does Chemical Cross-Linking with NHS Esters Reflect the Chemical Equilibrium of Protein-Protein Noncovalent Interactions in Solution?

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Author(s) Mädler, Stefanie, Seitz, Markus, Robinson, John, Zenobi, Renato
Publication Type Journal Items, Publication Status: Published
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Title Does Chemical Cross-Linking with NHS Esters Reflect the Chemical Equilibrium of Protein-Protein Noncovalent Interactions in Solution?
Author(s) Mädler, Stefanie
Seitz, Markus
Robinson, John
Zenobi, Renato
Journal or Series Title Journal of the American Society for Mass Spectrometry
Volume Number 21
Issue Number 10
Start Page 1775
End Page 1783
ISSN 1044-0305
Publisher Elsevier Science
Publication Place New York
Publication Date 2010
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 K-D range of 30 nM to >25 mu M, and (2) alpha-thrombin and basic pancreatic trypsin inhibitor (BPTI), a system that shows a buffer-dependent K-D value between 100 and 320 mu M. 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 K-D approximate to 25 mu M, 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 alpha-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 K-D >> 25 mu M.
DOI 10.1016/j.jasms.2010.06.016
Additional Notes Received 15 March 2010, Revised 20 June 2010, Accepted 20 June 2010, Available online 7 July 2010
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 03430
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NEBIS System Number 000541631
Source Database ID WOS-000282548800015
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  author = "M{\"{a}}dler, Stefanie and Seitz, Markus and Robinson, John and Zenobi, Renato",
  title = "{D}oes {C}hemical {C}ross-{L}inking with {N}{H}{S} {E}sters {R}eflect the {C}hemical {E}quilibrium of {P}rotein-{P}rotein {N}oncovalent {I}nteractions in {S}olution?",
  journal = "Journal of the American Society for Mass Spectrometry",
  year = 2010,
  volume = "21",
  number = "10",
  pages = "1775--1783",

E-Citations record created: Mon, 25 Oct 2010, 09:00:42 CET