Fast photochemical oxidation of proteins for comparing solvent-accessibility changes accompanying protein folding: Data processing and application to barstar |
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Affiliation: | 1. Donald Danforth Plant Science Center, Washington University, 975 N. Warson Rd. St. Louis, MO 63132, USA;2. AB Sciex, 500 Old Connecticut Path, Washington University, Framingham, MA 01701, USA;3. Department of Chemistry, Box 1134, Washington University, One Brookings Drive, St. Louis, MO 63130, USA |
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Abstract: | Mass spectrometry-based protein footprinting reveals regional and even amino-acid structural changes and fills the gap for many proteins and protein interactions that cannot be studied by X-ray crystallography or NMR spectroscopy. Hydroxyl radical-mediated labeling has proven to be particularly informative in this pursuit because many solvent-accessible residues can be labeled by OH in a protein or protein complex, thus providing more coverage than does specific amino-acid modifications. Finding all the OH-labeling sites requires LC/MS/MS analysis of a proteolyzed sample, but data processing is daunting without the help of automated software. We describe here a systematic means for achieving a comprehensive residue-resolved analysis of footprinting data in an efficient manner, utilizing software common to proteomics core laboratories. To demonstrate the method and the utility of OH-mediated labeling, we show that FPOP easily distinguishes the buried and exposed residues of barstar in its folded and unfolded states. This article is part of a Special Issue entitled: Mass spectrometry in structural biology. |
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