Modeling data from titration, amide H/D exchange, and mass spectrometry to obtain protein-ligand binding constants

• Published in: Journal of the American Society for Mass Spectrometry 2004-03, Vol.15 (3), p.388-397
• Main Authors: Zhu, Mei M, Rempel, Don L, Gross, Michael L
• Format: Article
• Language: English
• Subjects: Affinity; Amides - chemistry; Analytical, structural and metabolic biochemistry; Binding and carrier proteins; Binding sites; Biological and medical sciences; Calcium - metabolism; Calmodulin; Calmodulin - chemistry; Calmodulin - metabolism; Data acquisition; Deuterium; Deuterium Exchange Measurement; Differential equations; Exchanging; Fundamental and applied biological sciences. Psychology; Interaction parameters; Ligands; Mass spectrometry; Mathematical models; Modelling; Models, Molecular; Parameter uncertainty; Proteins; Proteins - chemistry; Proteins - metabolism; Regression analysis; Resampling; Scientific imaging; Spectrometry, Mass, Electrospray Ionization - methods; Spectroscopy; Thermodynamics; Titration; Titrimetry
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2003.11.007

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.