Critical Evaluation of Mass Spectrometric Measurement of Dissociation Constants

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Author(s) Mathur, Sonal, Badertscher, Martin, Scott, Michael, Zenobi, Renato
Publication Type Journal Items, Publication Status: Published
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Title Critical Evaluation of Mass Spectrometric Measurement of Dissociation Constants
Subtitle Accuracy and Cross-Validation against Surface Plasmon Resonance and Circular Dichroism for the Calmodulin-Melittin System
Author(s) Mathur, Sonal
Badertscher, Martin
Scott, Michael
Zenobi, Renato
Journal or Series Title Physical Chemistry Chemical Physics
Volume Number 9
Issue Number 47
Start Page 6187
End Page 6198
ISSN 1463-9076
Publisher The Royal Society of Chemistry
Publication Place Letchworth
Publication Date 2007
Abstract We present a comprehensive study for determining the binding affinity of a protein–ligand complex, using mass spectrometric methods. Mass spectrometry has been used to study noncovalent interactions for a number of years. However, the use of soft ionization mass spectrometry for quantitative analysis of noncovalently bound complexes is not widely accepted. This paper reports a comparison of MS methods against established methods such as surface plasmon resonance (SPR) and circular dichroism (CD) whose suitability for the quantitative assessment of noncovalent interactions is well known. ESI titration and MALDI-SUPREX were used as representative mass spectrometric methods for this work. We chose to study the calmodulin–melittin complex that presents three challenges: (i) it exhibits a high affinity (low nanomolar KD); (ii) complexes are formed only in the presence of a coactivator, calcium ions in this case; and (iii) the protein and the complex show a different ionization efficiency. Dissociation constants were obtained from each method for the selected system and compared thoroughly to elucidate pros and cons of the selected methodologies in terms of their ability for the determination of binding constants of protein–ligand complexes. ESI titration, SPR, CD and MALDI-SUPREX yielded KD values in the low nanomolar range that are in general agreement with an older value reported in the literature. We also critically evaluated the limitations in particular of the MS methods and the associated data evaluation procedures. We present an improved evaluation of SUPREX data, as well as a detailed error analysis for all methods used.
DOI 10.1039/b707946j
Additional Notes Received 24th May 2007
accepted 12th July 2007
first published as an advance article on the web 7th August 2007
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 02207
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NEBIS System Number 002005974
Source Database ID PP-34127
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  author = "Mathur, Sonal and Badertscher, Martin and Scott, Michael and Zenobi, Renato",
  title = "{C}ritical {E}valuation of {M}ass {S}pectrometric {M}easurement of {D}issociation {C}onstants: {A}ccuracy and {C}ross-{V}alidation against {S}urface {P}lasmon {R}esonance and {C}ircular {D}ichroism for the {C}almodulin-{M}elittin {S}ystem",
  journal = "Physical Chemistry Chemical Physics",
  year = 2007,
  volume = "9",
  number = "47",
  pages = "6187--6198",

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