Establishment And Application Of High-precision Large-scale Analysis Method Of Palmitoylated Protein

The S-palmitylation of proteins is an important post-translational modification,its dynamics and unique reversibility have endowed proteins with rich physiological functions.Palmitoylation has an important effect on virus infecting host cells.It affects host cell infection by affecting the location and aggregation of viral proteins on the cell membrane,which has been studied in influenza A viruses.Palmitoylated proteins are mainly distributed in brain tissues and regulate the function of neuronal proteins.The abnormal content of palmitoylated proteins is closely related to neurological diseases.As rabies is a malignant infectious disease with zoonosis,80%of the cases are cerebral rabies,which shows strong neurophagicity.The study of palmitoylation in rabies virus is of great significance for understanding the process of rabies virus infection.At present,research on palmitoylated proteins mainly relies on Acyl-Biotin Exchange(ABE).This method cuts the target protein by hydroxylamine(HA)to expose new thiol sites,then reacts the thiol reaction reagent with biotin.Finally,the streptavidin agarose gel beads were used to purify and enrich the biotinylated protein to detect palmitoylated protein.However,the disadvantage of this method is the high false positive rate and the inaccurate location of modification sites,which greatly limits the research and application of palmitoylated proteins.Our study improved the traditional ABE method,established a highly reliable method for the analysis of palmitoylated proteins,and successfully applied on a mice model of rabies.Finally,using bioinformatics to perform functional analysis of proteins with significant differences in the brain tissue of three groups of mice model,explore the relationship between the differential expression of proteins in rabies mice at different stages and disease development,and provide a certain experimental basis for furtherstudying the pathogenesis of rabies.In this experiment,we first studied the distribution of the protein.The samples were subjected to ultra high speed centrifugation during the extraction of mouse brain tissue protein,and ABE reactions were performed on the supernatant and the precipitate.The feasibility of the ABE method was verified by Western-Blot,and it was proved that palmitoylated proteins were mainly distributed in membrane proteins.Then compare the effect of disulfide reducing agent-TCEP on the experiment,and use Western-Blot to identify two groups of enriched proteins.It is proved that TCEP pretreatment can significantly reduce background interference and greatly reduce the false positive rate,which is an essential step in the experiment process.This experiment can also provides a method for rapid identification of the target protein without cleavage after enrichment,which provides an effective monitoring step before mass spectrometry detection and ensures the efficiency of mass spectrometry testing.Then,using this method to explore the dosage of agarose beads,it was found that 2?L agarose beads are sufficient to capture 100 ?g of the target protein.Finally,the established method was applied on a mice model of rabies,and palmitoylated proteins in the onset mice were significantly different from those in the latency and normal groups.Then use mass spectrometry to identify and analyze the protein in the brain tissue of mice model,and a total of 1868 proteins were identified,of which 283 were significantly different proteins,including the rabies virus main antigen G protein.Finally,bioinformatics was used to analyze the functional differences of the three groups of mice model.It was proved that the biologically significant proteins involved in the different stages of mice rabies were metabolic pathways,and the main molecular functions were catalytic activity and binding activity.It provides a theoretical basis for the research on the pathogenesis of zoonotic rabies.