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Article

H/D Exchange- and Mass Spectrometry-Based Strategy for the Thermodynamic Analysis of Protein−Ligand Binding

Supporting Info

Liangjie Tang,^† Erin D. Hopper,^† Yan Tong,^†^‡ Jack D. Sadowsky,^§ Kimberly J. Peterson,^§ Samuel H. Gellman,^§ and Michael C. Fitzgerald*^†

Department of Chemistry, Duke University, Durham, North Carolina 27708, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706

Anal. Chem., 2007, 79 (15), pp 5869–5877

DOI: 10.1021/ac0700777

Publication Date (Web): June 21, 2007

†

Duke University.

‡

Present address: McKinsey & Co., Shanghai 200020, China.

University of Wisconsin.

Corresponding author. Tel: 919-660-1547. Fax: 919-660-1605. E-mail: michael.c.fitzgerald@duke.edu.

Abstract

The equilibrium unfolding properties of four model protein systems were characterized using SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is an H/D exchange- and mass spectrometry-based technique for measuring the free energy (ΔG_f) and m-value (δΔG_f/δ[denaturant]) associated with the folding/unfolding reaction of a protein. The model proteins in this study (calmodulin, carbonic anhydrase II, RmlB, Bcl-x_L) were chosen to test the applicability of SUPREX to the thermodynamic analysis of larger (>15 kDa) or multidomain proteins. In the absence of ligand, ΔG_f and m-values for these proteins could not be evaluated using the conventional data acquisition and analysis methods previously established for SUPREX. However, ligand-bound forms of the proteins were amenable to conventional SUPREX analyses, and it was possible to evaluate reasonably accurate and precise binding free energies of selected ligands. In some cases, protein−ligand dissociation constants (K_d values) could also be ascertained. The SUPREX-derived binding free energies and K_d values evaluated here were in good agreement with those reported on the same complexes using other techniques.