A synthetic toolkit for the recombinant production of tyrosine phosphorylated proteins and peptides

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Project Summary Tyrosine phosphorylation (pTyr) is a post-translational modification that can regulate protein function and interac- tions. Tyrosine phosphorylation often becomes dysregulated in cancer and therefore, understanding the effect of phosphorylation on protein function and within biochemical networks will be paramount to identifying therapeutic interventions in cancer. Unfortunately, much of tyrosine phosphorylation is referred to as the “dark phosphopro- teome”, because we either cannot detect it or we do not understand what its role is in regulating proteins and networks. The dark phosphoproteome exists as a result of limitations in tools for testing and measuring pTyr within proteins and networks. In previous work, we developed a synthetic toolkit that has superior yields of phys- iologically relevant tyrosine phosphorylation on proteins of interest, compared to existing approaches. The goal of this work is to: 1) harden this technology for increased reproducibility and flexibility and 2) extend this technol- ogy to produce reagents, at a fraction of their current cost, for use in quantitative measurement and monitoring of phosphotyrosine sites in mass spectrometry-based workflows relevant to cancer progression and treatment response.