A Novel Method To Identify The Phosphorylation Sites Of RIP3 During Necroptosis With Biotin-labeling And Denatured Immunoprecipitation

Necroptosis is a type of cell death that is induced by TNF-? and is regulated,so it is related to many pathology and physiology process.Research about RIPS is always highly anticipated as it works as the key protein in the process of this kind of necrosis.When necroptosis happens,RIP3 would be activated by its upstream signaling protein RIP1 and one of the markers of this process is the phosphorylation of RIP3.Until now,only one functional phosphorylation site,Ser227,of RIP3 was found.Since the size of activated necrosome is relatively huge,it would always be in the insoluble part of cell lysate.So,these activated necrosome is always ignored for its insoluble characteristic in the former research.In order to reveal the important information in this part,we tried the new strategy to get and analyze activated necrosome from the insoluble part.The new way made use of the strong binding force that cannot be destroyed by strong detergent(SDS)between St:reptavidin and biotin,replacing natural immunoprecipitation(IP)with denatured IP to enrich biotin-labeled target proteins for mass spectrum(MS)analysis.There are two systems that could specifically label proteins with biotin:BirA in AVI-tag/BirA system could label AVI-tag of AVI-RIP3,while APEX2-tag system could label RIP3 and proteins spatial near to it through enzymatic reaction.We used these two ways to gain the potential phosphorylation sites and protein candidates related to RIP3.In this study,we optimize the experimental method to enrich and analyze information of the protein complex in the insoluble part.This novel method could be used to explore relatively huge protein complexes which were ignored for the insoluble property in the signaling pathway.