Abstract
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 Ca2+ 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.
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Published online February 7, 2004
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Zhu, M.M., Rempel, D.L. & Gross, M.L. Modeling data from titration, amide H/D exchange, and mass spectrometry to obtain protein-ligand binding constants. J Am Soc Mass Spectrom 15, 388–397 (2004). https://doi.org/10.1016/j.jasms.2003.11.007
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DOI: https://doi.org/10.1016/j.jasms.2003.11.007