Kanan Yogita, Al Ubaidi Muayyad R
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.
Curr Protoc Protein Sci. 2015 Apr 1;80:14.7.1-14.7.20. doi: 10.1002/0471140864.ps1407s80.
Tyrosine sulfation is a post-translational modification (PTM) where a sulfate group is added to a tyrosine moiety. This PTM is responsible for strengthening interaction between proteins. One of the drawbacks of studying this PTM is the lack of an antibody that can detect all tyrosine-sulfated proteins. In addition, due to the labile nature of the tyrosine sulfate, other techniques such as mass spectrometry cannot be used to study this PTM unless special modification procedures are used. This requires considerable skill and knowledge of mass spectrometry. This unit describes an in vitro technique that can be used to study tyrosine-sulfated proteins by radiolabeling the recombinant protein. The protein is then subject to barium hydroxide hydrolysis and thin-layer electrophoresis (TLE). Co-localization of radioactive tyrosine-sulfate with nonradioactive tyrosine sulfate standard spiked in before TLE analysis identifies a protein as tyrosine-sulfated protein. The advantage of this technique is that, it identifies all tyrosine-sulfated proteins without any bias and is the only technique that identifies the tyrosine sulfate residues in the protein.
酪氨酸硫酸化是一种翻译后修饰（PTM），其中硫酸基团被添加到酪氨酸部分。这种PTM负责加强蛋白质之间的相互作用。研究这种PTM的一个缺点是缺乏能够检测所有酪氨酸硫酸化蛋白的抗体。此外，由于酪氨酸硫酸盐的不稳定性质，除非使用特殊的修饰程序，否则质谱等其他技术不能用于研究这种PTM。这需要相当的质谱技术和知识。本单元描述了一种体外技术，可通过对重组蛋白进行放射性标记来研究酪氨酸硫酸化蛋白。然后将该蛋白进行氢氧化钡水解和薄层电泳（TLE）。在TLE分析之前加入的放射性酪氨酸硫酸盐与非放射性酪氨酸硫酸盐标准品的共定位可将一种蛋白鉴定为酪氨酸硫酸化蛋白。该技术的优点是，它能无偏差地鉴定所有酪氨酸硫酸化蛋白，并且是唯一能鉴定蛋白中酪氨酸硫酸化残基的技术。
