Chemical Proteomics-based Method To Quantitative Study UTP-binding Proteins In Human Cells

Nucleotide-binding proteins play important roles in a variety of biological processes.Although ATP-binding protein and GTP-binding protein have been well studied,there are still great challenges in the systematical study of UTP-binding proteins at the entire proteome level.In this article,we developed the UTP probe combined with the quantitative proteomics strategy based on stable isotope labeling by amino acids in cell culture(SILAC)to identify and quantitatively evaluate UTPbinding proteins in HEK 293 T cells.The main research contents are summarized as follows:(1)Synthesis,characterization and reactivity detection of UTP probes.First,the biotinylated amino acid precursor was coupled with UTP to synthesize UTP probe,and then the product was separated by high performance liquid chromatography.After characterization,the UTP probe was incubated with lysine to detect its reactivity.The results showed that the UTP probe can covalently label the UTP binding protein,indicating that it had high reactivity with the amino group of lysine.(2)A competitive strategy based on low or high concentration of UTP probes combined with SILAC-based quantitative proteomics strategy was used to identify potential UTP binding proteins.Taking advantage of the characteristic that the binding of specific proteins with probes do not depend on changes in concentration,a total of172 proteins were identified from HEK 293 T cell lysates,and some biotin-labeled peptide information was obtained.Using RUTP 10/1<2 as the standard to screen potential UTP targets,166 of them were finally identified as potential UTP-binding proteins,and it was found that they included some known UTP binding proteins,such as the heat shock protein HSP90.These results indicate that our method has a good ability to identify UTP binding proteins.(3)A competitive strategy based on UTP or UTP probes combined with SILACbased quantitative proteomics strategy was used to further evaluate the specificity of the target protein.The parent compound UTP was used as a competitor for competitive labeling with UTP probe,and then the labeling efficiency of the probe was analyzed through the identification results of mass spectrometry.Finally,a total of 303 proteins were quantified.According to the standard of RUTP/UTP probe<1,160 protein data was consistent with the requirements.Comprehensive analysis of the data of the two competition experiments,there were 71 proteins that match the above two conditions at the same time,and they were considered to be the most likely UTP binding proteins.Bioinformatics analysis showed that these UTP-binding protein candidates were involved in multiple biological processes.In summary,we used specific biotinylated UTP probes combined with a SILACbased quantitative proteomics workflow to conduct two types of competition experiments and identified more than 70 potential UTP binding proteins.They are involved in multiple biological processes,including translational elongation,nucleotide binding,and protein folding.Further characterizations of these UTPbinding proteins will improve our understanding about the biological functions of UTP in human cells.