Jeremiah Tipton - Sites and Extent of Selenomethionine Incorporation into Recombinant Cas6 Protein by Top-down and Bottom-up Proteomics with 14.5 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

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  Publication Details (including relevant citation   information):

  Wang, X.; Tipton, J.D.; Emmett, M.R.; Marshall,   A.G. Rapid Communications in Mass Spectrometry, 24 (16),   2386-2392, (2010)


  Selenomethionine-modified proteins can improve X-ray   crystallographic structural resolution by multi-wavelength   anomalous diffraction (MAD) phasing. However, the specificity and   extent of selenomethionine incorporation must first be assessed.   Bottom-up and top-down proteomics with a modified 14.5 T LTQ   Fourier transform ion cyclotron resonance mass spectrometer offer   a quick, accurate, and robust method to locate and quantify   selenomethionine incorporation after auxotrophic expression.   Selenomethionine (methionine with sulfur replaced by selenium)   has a different natural-abundance isotopic distribution and a   mass increase of 47.94 Da relative to wild-type methionine. Here,   both wild-type and selenomethionine-substituted forms of the Cas6   protein containing 'clustered regularly interspaced short   palindromic repeats' (CRISPRs) were expressed and purified.   Comparative bottom-up and top-down proteomics confirmed that all   six methionines were fully replaced by selenomethionines in   Se-Cas6. Copyright © 2010 John Wiley & Sons, Ltd.

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