Determination of dissociation constants for protein-ligand complexes by electrospray ionization mass spectrometry.
A fully automated biophysical assay based on electrospray ionization mass spectrometry (ESI-MS) for the determination of the dissociation constants (KD) between soluble proteins and low molecular mass ligands is presented. The method can be applied to systems where the relative MS response of the protein and the protein-ligand complexes do not reflect relative concentrations. Thus, the employed approach enables the use of both electrostatically and nonpolar bound complexes. The dynamic range is wider than for most biological assays, which facilitates the process of establishing a structure-activity relationship. This fully automated ESI-MS assay is now routinely used for ligand screening. The entire procedure is described in detail using hGHbp, a 25-kDa extracellular soluble domain of the human growth hormone receptor, as a model protein.
Tjernberg
Agneta
A
Department of Structural Chemistry, Biovitrum AB, SE-112 76 Stockholm, Sweden. agneta.tjernberg@biovitrum.com
Carnö
Sofi
S
Oliv
Frida
F
Benkestock
Kurt
K
Edlund
Per-Olof
PO
Griffiths
William J
WJ
Hallén
Dan
D
D006801:Humans; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D011506:Proteins / Q000737:chemistry*; D011986:Receptors, Somatotropin / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D055672:Static Electricity
D008024:Ligands; D011506:Proteins; D011986:Receptors, Somatotropin