Analytica Chimica Acta 496 (2003) 225–232 The accuracy and precision of a new H/D exchange- and mass spectrometry-based technique for measuring the thermodynamic stability of proteins Kendall D. Powell, Michael Z. Wang, Peter Silinski, Liyuan Ma, Thomas E. Wales, Susie Y. Dai, Anne H. Warner, Xiaoye Yang, Michael C. Fitzgerald ∗ Department of Chemistry, Box 90346, Duke University, Durham, NC 27708-0346, USA Received 18 October 2002; accepted 18 October 2002 Abstract Recently, we developed a new method for measuring the thermodynamic stability of proteins. The method, termed Stability of Unpurified Proteins from Rates of H/D Exchange (SUPREX), utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and exploits the H/D exchange properties of proteins to determine folding free energies (i.e. G ◦ f values) for proteins. Here we report on the SUPREX analysis of seven new model proteins. The results of these analyses and the results of previously reported SUPREX analysis on seven additional proteins are used to assess the accuracy and precision of the SUPREX technique for measuring G ◦ f values. We find that the accuracy of the SUPREX technique for measuring the G ◦ f values of proteins is on the order of 20% and precision (relative standard deviation) of the technique is on the order 10%. These measures of accuracy and precision are comparable to those of conventional methods. © 2003 Elsevier B.V. All rights reserved. Keywords: Accuracy and precision; H/D exchange; Thermodynamic stability 1. Introduction Thermodynamic measurements of protein stability (i.e. measurements of G ◦ f ) are widely used in the field of protein biochemistry. Such measurements are essential in mutational studies of protein folding and stability; and they are commonly used to investigate the strength of protein–ligand binding interactions. A variety of different methods have been utilized for making G ◦ f measurements on proteins. Methods have been developed that exploit such techniques ∗ Corresponding author. Tel.: +1-919-660-1547; fax: +1-919-660-1605. E-mail address: mfitz@chem.duke.edu (M.C. Fitzgerald). as nuclear magnetic resonance (NMR) spectroscopy, calorimetry, and optical techniques such as fluo- rescence and circular dichroism (CD) spectroscopy [1–4]. Recently, we developed a new method for making G ◦ f measurements on proteins that utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and exploits the H/D exchange properties of proteins [5–7]. Our new method, which we have termed Stability of Unpurified Proteins from Rates of H/D Exchange (SUPREX), has several ex- perimental advantages over conventional methods for making G ◦ f measurements. These advantages include: the ability to make measurements on pi- comole quantities of protein; the ability to make measurements on unpurified protein samples; and the 0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0003-2670(03)01002-X