Modeling data from titration, amide H/D exchange, and mass spectrometry to obtain protein-ligand binding constants.
We recently reported a new method for quantification of protein-ligand interaction by mass spectrometry, titration and H/D exchange (PLIMSTEX) for determining the binding stoichiometry and affinity of a wide range of protein-ligand interactions. Here we describe the method for analyzing the PLIMSTEX titration curves and evaluate the effect of various models on the precision and accuracy for determining binding constants using H/D exchange and a titration. The titration data were fitted using a 1:n protein:ligand sequential binding model, where n is the number of binding sites for the same ligand. An ordinary differential equation was used for the first time in calculating the free ligand concentration from the total ligand concentration. A nonlinear least squares regression method was applied to minimize the error between the calculated and the experimentally measured deuterium shift by varying the unknown parameters. A resampling method and second-order statistics were used to evaluate the uncertainties of the fitting parameters. The interaction of intestinal fatty-acid-binding protein (IFABP) with a fatty-acid carboxylate and that of calmodulin with Ca(2+) are used as two tests. The modeling process described here not only is a new tool for analyzing H/D exchange data acquired by ESI-MS, but also possesses novel aspects in modeling experimental titration data to determine the affinity of ligand binding.
Department of Chemistry, Washington University, St, Louis, Missouri 63130, USA.
Mei M
MM
Zhu
Don L
DL
Rempel
Michael L
ML
Gross
D000577:Amides / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D008024:Ligands; D008958:Models, Molecular; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013816:Thermodynamics; D017186:Titrimetry
D000577:Amides; D002147:Calmodulin; D008024:Ligands; D011506:Proteins; D002118:Calcium