Activity Based Probes For Protease Profiling In Hela Cells

With its ability to bridge the gap between the genome sequence and cellular functionalities of proteins,activity-based proteomics has emerged as a major tool for the high-throughput identification and characterization of proteins.In complex system of the life,the big family,protease is a major player in proteolysis which has closely related to disease.In order to establish complete proteases proteomic profiling and find the corresponding tumor biomarks,in this thesis,we fouced on proteases in cervical cancers with assistance of activity based probes.Bascially,crude lysates are treated with various activity-based probes that target active proteases.The interested proteases were separated with affinity pull down and identified by LC-MS/MS.So this thesis includes the following parts:First,according to the literature,13 activity based probes based on a quinolimine methide reactive group were successfully synthesized containing a)different amino acids as substrates in P1 position to be recognized and hydrolyzed by proteases;b)a terminal alkyne as a click group.All the compounds were with the structural characterization of ¹H NMR,¹³C NMR and LC-Mass.Second,with all the probes in hand,labeling experiments were performed against nine pure commercial available proteases,including trypsin,endoproteasease,proteasease,penicillin amidasde,pronase,papain,themolysin,collagerase,acylase 1,trypsin.With optimized conditions,the degree of labeling in each protease by these probes is correspoinding to their known P1 position substrate specificity.Furthermore,unique activity-based fingerprint profiles of these nine proteases were generated.All these results also strongly supported these probes are feasible to analyze more complicated protease proteomics.Third,we next investigated the protease profiling in HeLa cell lysate with these P1 postion based probes to find the known and unknown proteases.With optimized conditions,the results showed that QMAc-M,QMAc-K and QMAc-L have showed higher labeling efficiency of cell lysate,which indicated that proteases with Met,Lys and Leu cleavage ability showed related high amount and activity.Cell imaging was conducted to check their localization and cell permeability of there probes.Labeled proteomics were enriched with neutravidin affinity pull down approached and LC-MS/MS will be further applied to understand the targets of labeled proteases.In conclusion,we have demonstrated that the 13 probes has been successfully designed,synthesized and performed in both pure proteases and HeLa cell lysate.This approach has been approved the detections of different classes of proteasese on the basis of their activities and substrate specificities.In the future work,we will continue to focus on the utilities in complex proteomics research to investigate novel protease based cancer biomarker.