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

Xu Wang, Jeremiah D. Tipton, Mark Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 naturalabundance 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.

Original languageEnglish (US)
Pages (from-to)2386-2392
Number of pages7
JournalRapid Communications in Mass Spectrometry
Volume24
Issue number16
DOIs
StatePublished - Aug 30 2010
Externally publishedYes

Fingerprint

Selenomethionine
Cyclotrons
Cyclotron resonance
Fourier Analysis
Recombinant Proteins
Proteomics
Mass spectrometry
Mass Spectrometry
Fourier transforms
Ions
Methionine
Clustered Regularly Interspaced Short Palindromic Repeats
Mass spectrometers
Selenium
Sulfur
Proteins
Diffraction
X-Rays
X rays
Wavelength

ASJC Scopus subject areas

  • Spectroscopy
  • Analytical Chemistry
  • Organic Chemistry
  • Medicine(all)

Cite this

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title = "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",
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 naturalabundance 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.",
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T1 - 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

AU - Wang, Xu

AU - Tipton, Jeremiah D.

AU - Emmett, Mark

AU - Marshall, Alan G.

PY - 2010/8/30

Y1 - 2010/8/30

N2 - 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 naturalabundance 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.

AB - 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 naturalabundance 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.

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