Quantitative Chemical Proteomics-based Analysis Of CTP-Binding Proteins In Human Cells

Cytidine triphosphate(CTP)is an essential nucleotide for nucleic acid biosynthesis and protein glycosylation.In addition,it also plays a vital role in membrane phospholipid synthesis and cell signal transmission.Studies have shown that CTP can act as a ligand to regulate the activity of its target proteins in many biological processes.However,it is still challenging to identify CTP binding proteins at proteome-wide.In this study,a CTP acyl affinity probe was developed and combined with the quantitative proteomics method of stable isotope labeling by amino acids in cell culture(SILAC)to capture,identify and quantify CTP binding proteins in human cells.The main contents of this thesis are summarized as follows:(1)Design and Synthesis of CTP acyl affinity probe.Activity-based protein profiling(ABPP)has the advantage of reflecting protein activity and binding sites.In this work,a simple two-step method was used to synthesize a CTP acyl affinity probe:CTP was used as a probe warhead to bind the target protein,γ-aminobutyric acid was used as an intermediate to reduce steric hindrance,and biotin was used as a label for subsequent affinity purification.Substances were then separated from the crude product by high performance liquid chromatography(HPLC).Mass spectrometry showed that the m/z of the product was 793.1,which was consistent with the CTP acyl affinity probe,proving that the probe was successfully synthesized.(2)Different concentrations of probe coupled with SILAC technology were used to identify CTP binding proteins.First,HEK 293 T cells were labeled with stable isotopes.Subsequently,different concentrations of CTP acyl affinity probe(10μM/100 μM)were conjugated with light-or heavy-labeled proteomes respectively to make the probe bind to the target proteins in the natural cell environment.After incubation,streptavidin gel beads were used to pull down the biotin-labeled proteins,and analyzed by liquid chromatography tandem mass spectrometry(LC-MS/MS)after trypsin digestion.LC-MS/MS identified a total of 575 potential CTP-binding proteins.These included known CTP binding proteins,CTP synthase I(CTPS1)and members of the heat shock protein family(HSP90s),which proves that this method can be used to identify CTP binding proteins.(3)Free CTP competition experiment combined with SILAC technology to verify the credibility of potential CTP binding proteins.If the candidate protein is indeed a CTP binding protein,the competition of CTP will reduce the number of proteins pulled down by the probe.Based on this principle,free CTP was added to compete before incubating the probe with the proteome,and then probe was added to label.Then the protein mixture was purified with streptavidin and analyzed by LC-MS/MS after digestion.A total of 129 potential CTP binding proteins were identified.Combining the two sets of experimental data,we identified a total of 90 proteins that meet the two screening criteria at the same time,and we identified them as CTP binding proteins.These included a part of the known CTP binding protein and a part of unknown CTP binding protein.Bioinformatics analysis showed that these CTP binding proteins are involved in a variety of cellular activities,including protein folding and cell adhesion.This work lays the foundation for the in-depth study of CTP binding proteins.