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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
Author(s) -
Wang Xu,
Tipton Jeremiah D.,
Emmett Mark R.,
Marshall Alan G.
Publication year - 2010
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4655
Subject(s) - chemistry , fourier transform ion cyclotron resonance , selenium , mass spectrometry , proteomics , top down proteomics , methionine , ion cyclotron resonance , analytical chemistry (journal) , sulfur , ion , crystallography , chromatography , biochemistry , tandem mass spectrometry , protein mass spectrometry , amino acid , cyclotron , organic chemistry , gene
Abstract 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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