Proteomic Analysis Of Mouse Endogenous UFMylation

[Objective] UFMylation is a post-translational modification of proteins,which plays a vital role in cell development and maintaining tissue homeostasis.The research team conducted large-scale proteomic analysis of liver tissue of C57 mice,combined with homemade UFM1 antibody,and identified UFMylated proteins and modified sites by high-resolution mass spectrometry.This large-scale identification of endogenous UFMylated proteins provides more experimental basis for people to study the function and mechanism of UFMylation.[Methods] 1.Design UFM1 antibody according to the characteristics of UFM1.2.Verify the effectiveness and specificity of self-made UFM1 antibody by IP(immunoprecipitation)and WB(immunoblotting).3.Pretreatment of C57 mouse liver tissues before mass spectrometry,including lysis,enzymatic hydrolysis and IP(immunoprecipitation).4.Proteomics analysis of the data.5.Verify UFM modified protein.[Results] 1.Compared with commercial UFM1 antibodies,homemade UFM1 antibodies have advantages in terms of specificity and effectiveness.2.A total of 203 modified sites of 178 UFMylated proteins were identified by high resolution mass spectrometry.3.RPL26,a known UFMylated protein,was identified in the database,and the modification sites K132 and K134 are in line with the report.4.Bioinformatics analysis shows that UFMylation is not only localized in the endoplasmic reticulum,but also abundant in mitochondria,which maybe be closely related to the redox process in cell metabolism.5.The research group propose a "ExxxK" conservative motif based on the laws of UFMylated proteins and sites.[Conclusion]:1.Designed and prepared antibodies capable of enriching UFM1 modified peptides,which can identify UFMylated sites on a large scale at the tissue level.2.UFMylation may be related to redox metabolism,and there may be conservative motifs at the modification site of UFM1.