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

J Mol Recognit. Jul-Aug 2009;22(4):319-29. doi: 10.1002/jmr.951.


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 al., 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 microM. 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 approximately 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Calorimetry / methods*
  • Carbonic Anhydrase I / metabolism*
  • Ethoxzolamide / metabolism
  • Humans
  • Kinetics
  • Ligands
  • Mass Spectrometry*
  • Protein Binding
  • Reproducibility of Results
  • Spectrometry, Mass, Electrospray Ionization
  • Staining and Labeling / methods*
  • Surface Plasmon Resonance*
  • Time Factors
  • Titrimetry


  • Ligands
  • Carbonic Anhydrase I
  • Ethoxzolamide