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.
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.
Wang
Xu
X
Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, Florida 32306, USA.
Tipton
Jeremiah D
JD
Emmett
Mark R
MR
Marshall
Alan G
AG
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D012645:Selenomethionine / Q000737:chemistry* / Q000378:metabolism
D019843:Archaeal Proteins; D011994:Recombinant Proteins; C095508:rCAS protein, recombinant; D012645:Selenomethionine; D004722:Endoribonucleases