Label-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry

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Author(s) Jecklin, Matthias C., Schauer, Stefan, Dumelin, Christoph E., Zenobi, Renato
Publication Type Journal Items, Publication Status: Published
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Title Label-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry
Author(s) Jecklin, Matthias C.
Schauer, Stefan
Dumelin, Christoph E.
Zenobi, Renato
Journal or Series Title Journal of molecular recognition
Volume Number 22
Issue Number 4
Start Page 319
End Page 329
ISSN 0952-3499
1099-1352
Publisher Wiley
Publication Place Chichester
Publication Date 2009
Keyword(s) nanoelectrospray mass spectrometry
noncovalent complexes
carbonic anhydrase
isothermal titration calorimetry
surface plasmon resonance
Abstract We performed a systematic comparison of three label-free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. The performance of (1) nanoelectrospray ionization mass spectrometry (nESI-MS), (2) surface plasmon resonance (SPR), and (3) isothermal titration calorimetry (ITC) was compared for the determination of dissociation constants (K-D). The model system studied for this purpose was the human carbonic anhydrase I (hCAI) with eight known and well characterized sulfonamide inhibitors (Krishnamurthy et at., Chem. Rev. 2008,108:946-1051). The binding affinities of the inhibitors chosen vary by more than four orders of magnitude e.g., the K-D value determined for ethoxzolamide by nESI-MS was 5 +/- 1 nM and the K-D value for sulfanilamide was 145.7 +/- 10.0 mu M. The agreement of the determined K-D values by the three methods investigated was excellent for ethoxzolamide and benzenesulfonamide (variation with experimental error), good for acetazolamide and 4-carboxybenzenesulfonamide (variation by similar to one order of magnitude), but poor for others e.g., sulpiride. The accuracies of the K-D values are determined, and advantages and drawbacks of the individual methods are discussed. Moreover, we critically evaluate the three examined methods in terms of ease of the measurement, sample consumption, time requirement, and discuss their limitations.
DOI 10.1002/jmr.951
Additional Notes Received 20 January 2009, Revised 10 March 2009, Accepted 10 March 2009, Published online 16 April 2009
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 03430
02207
Organisational Unit(s)
NEBIS System Number 000474824
Source Database ID PP-49142
PP-55225
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@article{Jckln2009,
  author = "Jecklin, Matthias C. and Schauer, Stefan and Dumelin, Christoph E. and Zenobi, Renato",
  title = "{L}abel-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry",
  journal = "Journal of molecular recognition",
  year = 2009,
  volume = "22",
  number = "4",
  pages = "319--329",
}


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