Identification Of Tyrosine Phosphorylation Binding Proteins By Photoaffinity Peptide Probe Library

Cellular signal transduction is often achieved through protein-protein dynamic interactions.Once a signal is terminated,the interacting signal proteins are separated in time to return the cell to a basic state ready to respond to the next stimulus.The key steps of these dynamic proteins are performed by modularized domains to affinity recognition of structures with specific sequence characteristics.Tyrosine Phosphorylation（pY）as a special signal docking sites,mediated the important signal transduction pathways,closely connected with the development of human disease.Current studies indicate that thousands of tyrosine phosphorylation sites are expected to exist in human cells,and their interaction network with specific recognition domains（e.g.,SH2）is not very clear,which seriously restricts the development of this field.However,because tyrosine phosphorylation and recognition protein abundance are often very low,their mediated protein interactions are weak and often instantaneous,so their interaction analysis is very challenging.In this paper,GST-SRC_SH2 protein was constructed and expressed to simulate the key structural domain of tyrosine phosphorylation binding protein reaction,and a tyrosine phosphorylation photoaffinity probe was designed and prepared by taking SHC1-pY³¹⁷ as an example,for the strong and specific enrichment of its target protein.We first optimized the system required for this type of probe reaction,and our results show that the developed probe can highly selectively capture specific binding target proteins even in complex environments.According to the design of the first photocrosslinked affinity probe,we hope to further expand the enrichment range of the target protein on the basis of its specificity and specificity.Therefore,on the basis of the introduction of combinatorial library of peptide ligands（CPLL）technology,build the tyrosine phosphorylation of combination light affinity probe library,and then based on mass spectrometry and proteomics technology,24 proteins were identified with specific protein tyrosine phosphorylation in combination with SH2 domain structure,compared with previously reported methods,this has better enrichment effect.In addition,we also found some recognition proteins that have not been reported but have potential binding capacity for pY.With the help of biochemical means,quantitative proteomics technology and structural analysis,we verified the specific recognition ability of PKN2 on pY.Our experimental results show that this method can not only characterize the dynamic interaction between tyrosine phosphorylation and the SH2domain,but also can be used to explore and excavate the unknown protein specific recognition proteins after translation modification.