Measurements of Protein Stability by H/D Exchange and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using Picomoles of Material

Abstract

Recently, we reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique, termed SUPREX, that can be used to measure the thermodynamic stability of a protein (Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8296-8301). In the work described here, we report on our efforts to optimize the sensitivity of SUPREX analyses. We describe a new sample handling protocol for SUPREX that involves the use of batch chromatography methods with reversed-phase chromatographic media for the microconcentration and desalting of SUPREX samples. Using ribonuclease A as a model protein system, we demonstrate that our new protocol permits the SUPREX analysis of as little as 10 pmol of protein. This amount of protein is 100-fold less than the amount of material required in our initial SUPREX protocol, and it is 1-2 orders of magnitude less than the amount of material required in conventional spectroscopy-based methods for measuring the thermodynamic stability of a protein.

Throughput and efficiency of a mass spectrometry-based screening assay for protein—Ligand binding detection

“…The conditions of the rescreening experiment were similar to those of the initial screening, except that C 18 ZipTips (Millipore, Billerica, MA) were used to concentrate and desalt the samples before single-point SUPREX analysis. The incorporation of ZipTips into the SUPREX protocol has been reported previously [25]. Briefly, this process involves quenching the H/D exchange reaction with 1 L of a 10% TFA solution and extracting the proteins from the H/D exchange buffers using ZipTips that were preequilibrated according to the manufacturer's instructions.…”

Studies of biomolecular conformations and conformational dynamics by mass spectrometry

“…Studies of protein equilibrium unfolding induced by chaotropes usually require extensive sample clean-up and thus cannot be carried out ''on-line,'' although Powell et al have managed successfully to measure protein stability by urea-induced unfolding with HDX MALDI MS (Powell & Fitzgerald, 2001;Powell, Wales, & Fitzgerald, 2002). Thermal denaturation is another way to study protein equilibrium unfolding that can be implemented with an ''on-line'' experimental set-up, as demonstrated by the Deinzer group (Maier, Schimerlik, & Deinzer, 1999).…”

Thermodynamic stability measurements on multimeric proteins using a new H/D exchange‐ and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry‐based method

“…We recently reported a new H/D exchange-and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that can be used to quantitate the thermodynamic stability of proteins (Ghaemmaghami et al 2000). In contrast to conventional methods, the SUPREX technique can be used to quantitate the stability of mg to ng quantities of both purified and unpurified proteins (Powell and Fitzgerald 2001). In SUPREX, protein samples are subjected to H/D exchange by dilution into a series of deuterated exchange buffers containing different concentrations of a chemical denaturant such as guanidinium chloride (GdmCl).…”

“…Initial reports on SUPREX have included experiments with ribonuclease A, maltose binding protein, and eight variants of a monomeric repressor construct (Ghaemmaghami et al 2000;Ghaemmaghami and Oas 2001;Powell and Fitzgerald 2001). The data obtained in these previous reports indicate that the SUPREX technique can be used to determine folding free energies of monomeric proteins with the precision of conventional techniques (typically < 5%).…”