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

摘要

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.