Site-specific incorporation of phosphotyrosine using an expanded genetic code

Christian Hoppmann, Allison Wong, Bing Yang, Shuwei Li, Tony Hunter, Kevan M. Shokat, Lei Wang

Access to phosphoproteins with stoichiometric and site-specific phosphorylation status is key to understanding the role of protein phosphorylation. Here we report an efficient method to generate pure, active phosphotyrosine-containing proteins by genetically encoding a stable phosphotyrosine analog that is convertible to native phosphotyrosine. We demonstrate its general compatibility with proteins of various sizes, phosphotyrosine sites and functions, and reveal a possible role of tyrosine phosphorylation in negative regulation of ubiquitination.

The MS analysis was carried out using Advion Expression® CMS ESI.