Objective:In mouse embryonic stem cells(mESC),proximity labeling technology based on engineered ascorbate peroxidase(APEX2)and mass spectrometry were used to identify nucleolar related proteins. Methods:An APEX2-FBL overexpression stable cell line was constructed,biotin-labeled protein was obtained by APEX2 proximity labeling technology,the biotin-labeled protein was enriche with magnetic beads coated with streptavidin,and then liquid chromatography-tandem mass spectrometry(LC-MS/MS),and bioinformatics analysis was used to screen differential proteins. Results:Highly expressed APEX2-FBL stable cell lines were suuessfully constructed;Western blot results showed that a large number of biotin-labeled proteins were obtained by APEX2 proximity labeling technology;837 differential proteins were identified by mass spectrometry,and the biological processes were mostly related to nucleoli. Conclusion:The use of adjacent biotin labeling technology combined with mass spectrometry is a feasible method to identify nucleolar related proteins.