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

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Powell, KD; Fitzgerald, MC

Published Date

  • July 2001

Published In

Volume / Issue

  • 73 / 14

Start / End Page

  • 3300 - 3304

PubMed ID

  • 11476229

Pubmed Central ID

  • 11476229

Electronic International Standard Serial Number (EISSN)

  • 1520-6882

International Standard Serial Number (ISSN)

  • 0003-2700

Digital Object Identifier (DOI)

  • 10.1021/ac0100805

Language

  • eng