Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, Florida 32306, USA.
Rapid Commun Mass Spectrom. 2010 Aug 30;24(16):2386-92. doi: 10.1002/rcm.4655.
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.
硒代蛋氨酸修饰的蛋白质可以通过多波长异常衍射（MAD）相位分析提高 X 射线晶体学结构分辨率。然而，必须首先评估硒代蛋氨酸的特异性和掺入程度。带有改良的 14.5 T LTQ 傅里叶变换离子回旋共振质谱仪的自下而上和自上而下的蛋白质组学为在营养缺陷型表达后定位和定量硒代蛋氨酸掺入提供了一种快速、准确和强大的方法。硒代蛋氨酸（硫被硒取代的蛋氨酸）的天然丰度同位素分布不同，相对于野生型蛋氨酸的质量增加了 47.94 Da。在这里，含有“成簇规律间隔短回文重复”（CRISPRs）的 Cas6 蛋白的野生型和硒代蛋氨酸取代形式都被表达和纯化。比较自下而上和自上而下的蛋白质组学证实，Se-Cas6 中的所有六个蛋氨酸都被硒代蛋氨酸完全取代。
