Research On A New Method For Protein Arginine Methylation Analysis

Post-translational modification(PTM)of protein is the sub-molecular level modification of protein and polypeptide molecules at specific site,including phosphorylation,methylation,ubiquitination,glycosylation,and lipidation etc.PTM make the structure of protein more complex and diverse,function more perfect and specific,and regulation more delicate and delicate.It plays a very important role in the execution of normal physiological functions of living organism.Therefore,it became the representative of epigenetic research and protein functional group in post-genome era.At the same time,PTM is closely related to the regulation of cell metabolism,abnormal modification can cause dysfunction,including diabetes,cancer and metabolic syndrome are closely related to abnormal post-translational modification.Arginine methylation is a common post-translational modification catalyzed by the protein arginine methyltransferase family(PRMT),which is related to histone regulation,m RNA splicing,m RNA translation and cell signal transduction Such functions are closely related and have attracted widespread attention from biomedical researchers in recent years.However,arginine methylation modification is a modification of the protein at the sub-molecular level,and this type of modification does not change the charge of the modified protein,which puts forward higher requirement for the identification of methylation modification and protein interaction technology.Therefore,the identification of arginine methylation modification and the elucidation of the interaction between PTM and other protein,especially the study of the corresponding demethylase and the balance and regulation mechanism between the two,are of vital importance to biomedical research.At present,although many techniques can be used to study arginine methylation modification,the sub-molecular level modification on protein often show low abundance and dynamic change,which can only mediate weak and short-lived interaction,looking for arginine weak protein interaction mediated by methylation modification are challenging.This thesis focus on the identification of protein-protein interaction(PPI)and its status mediated by arginine methylation,we have developed two novel analytical technique at the omics level and the biochemical molecular level,respectively.In the first part,we developed a technology for capturing weakly interacting protein based on photo-affinity covalent linkage.Photo-affinity magnetic bead(PAMB)has been prepared using photo-affinity connection technology and applied to protein capture system to convert weak "protein-protein" interactions into covalent linkage,thereby identifying the captured protein.This study provides a capture magnetic bead and a new assay method for PPI-mediated protein modification network(such as PTM),which can be used to analyze the interaction proteomic of protein arginine methylation.The second part is the electrochemical detection of the methylation status of protein arginine based on the Sakaguchi reaction.We propose a direct and sensitive electrochemical analysis method to identify the methylation status of arginine.This method combines electrochemical technology with arginine-specific Sakaguchi reaction reaction,which is highly selective and sensitive.The electrochemical signal,developed a highly selective and simple electrochemical detection technology.This determination method provides a convenient technical method for detecting the methylation status of protein arginine,facilitates the subsequent screening of arginine methyltransferase inhibitors,and help to screen protein arginine methylase and demethylase.In summary,we have proposed new methods to analyze the weakly interacted proteins of arginine methylation sites,and the identification of arginine methylation status,thereby strengthening the studies of protein arginine methyltransferase,arginine demethylase and interacted proteins for the advancement of arginine methylation research.