Method Development For The Detection Of Newly Synthesized Proteins Via Bioorthogonal Chemistry

The method of using biological orthogonal chemistry for specific labeling of biomolecules(such as proteins)in native cellular is becoming increasingly mature.But Most of the work in this field is focused on the whole study of the newly synthesized proteins under certain conditions.That is,the object and the target molecule are a set of protein mixtures(proteome).And the samples to be tested were mostly cell lysis or tissue homogenate.However,there is no mature methods for the new generation of target protein molecules.Therefore,it is of great significance for specific labeling of proteins in living systems,the study of newly synthesized proteins,and the characterization of protein metabolism.In this study,a method for the detection of newly synthesized protein was developed,in which the Raw264.7 cells was treated by lipopolysaccharide(Lipopolysaccharide,LPS)to generate tumor necrosis factor alpha.A variety of experimental parameters including the concentration of LPS and the duration of LPS treatment were investigated by measuring the LPS stimulated TNF-alpha from Raw264.7 cells.The optimal experimental conditions were determined: Raw264.7 cells were cultured in Met-free DMEM containing 1% fetal bovine serum(FBS),and then treated for 4h with LPS at concentration of 10ng/m L in presence of Non-natural amino acid(Azidohomoalanine,AHA).The biotin tags were introduced into proteins containing AHA by copper-catalyzed alkyne-azide cycloaddition(Cu AAC).Then TNF-alpha was specifically captured by the antibody adsorbed on the solid support,on which the biotin tag could be detected by streptavidin coupled with horseradish peroxidase(HRP).Thus,the purpose of detecting specific newly synthesized protein(TNF-alpha)is achieved.However,TNF-alpha is secreted protein.The content of TNF-alpha in cell lysis cannot accurately represent its new generation.So,we developed a gel click chemistry.Use SDS-PAGE electrophoresis or Two-dimensional electrophoresis to remove free AHA molecules in samples.Then the proteins in gel were labeled with biotin by click chemistry.On which the biotin tag could be detected by streptavidin coupled with HRP.By scanning the total signal strength of a protein point and the signal strength of HRP,the qualitative analysis of a newly synthesized protein can be performed.Although,studies have shown that the AHA no toxicity to cells,the addition of foreign substances into cells causes physiological changes within cells.So the samples labeled with AHA and Met were analyzed by liquid chromatography-mass spectrometry,we found 87 up-regulated proteins and 54 down-regulated proteins.This study provided a method for the detection of a specific newly synthesized protein,characterization of specific protein turnover,and exploration of tumor markers.