Measurements of protein stability by H/D exchange and matrix-assisted laser desorption ionization mass spectrometry using picomoles of material

Recently, we reported on a new H/D exchange- and matrix-assisted laser deso rption/ionization (MALDI)-based technique, termed SUPREX, that can be used to measure the thermodynamic stability of a protein (Ghaemmaghami, S.; Fitz gerald, 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 sen sitivity 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 SU PREX samples, Using ribonuclease A as a model protein system, we demonstrat e 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 ma terial required in our initial SUPREX protocol, and it is 1-2 orders of mag nitude less than the amount of material required in conventional spectrosco py-based methods for measuring the thermodynamic stability of a protein.