Application Of Protein Cysteine And Their Oxidation Modification Probe In Chemical Proteome Analysis

Cysteines are unique among all protein-coding amino acids due to their inherent high nucleophilicity.The sulfur atoms of protein cysteines can assume a number of different redox states and thus exhibit a wide range of chemical reactivity.They can be readily affected by a wide range of redox mechanisms,resulting in various forms of oxidative post-translational modifications,such as cysteine sulfenic acid（-SOH）,sulfinic acid（-SO₂H）,sulfonic acid（-SO₃H）,S-sulfhydryl persulfide（-SSH）,S-nitrosothiol（-SNO）,and disulfide bonds（-S-S-）.Like many other post-translational modifications（e.g.tyrosine phosphorylation）,redox-dynamic modifications of cysteines can also reversibly alter protein structure and function,which has emerged as a key cellular signaling mechanism involved in a variety of redox-dependent life processes such as adipogenic thermogenesis,leukocyte recruitment,neuronal excitation and DNA damage repair.Thus,protein cysteines and their oxidative modifications play very crucial roles in various life processes.In recent years,chemical biology researchers have developed a variety of chemical probes for protein cysteines and their oxidative modifications and corresponding chemical proteomic analysis strategies,which have been applied to study specific redox biological mechanisms,develop covalent ligands,identify drug/natural product modification sites,and probe disease markers,among other fields.However,the currently reported labeled probes for protein cysteines and their oxidative post-translational modifications have their own advantages and disadvantages,and often cannot simultaneously take into account the advantages of high specificity,high reaction rate,and compatibility with mass spectrometry,and need to be combined with different chemical proteomic analysis strategies to obtain more satisfactory results when solving specific problems.The main work of this thesis is as follows:1,Selenium-containing probes designed and synthesized based on Se-N and Se-Se bonds as covalent reaction warheads,and the selenium probes showed high specificity and reactivity to cysteine thiols on small molecules and purified proteins,and exhibited good competition labeling in Gel-ABPP.More than 6000 candidate thiol proteins were identified using selenium probes combined with chemical proteomic approaches.These proteins involve a large number of redox homeostasis-related proteins,which will provide new tools for probing cellular redox homeostasis signaling and other aspects.2.The cysteine sites that can be labeled by the selenium probe were identified using a chemical proteomic approach.The peptides labeled by the selenium probe were highlighted to exhibit distinct selenium isotope signature peaks in primary and secondary mass spectra.This unique isotope signature peak could provide compelling evidence for the identification of redox-regulated proteins and other target peptides.Analysis of post-translational modifications（PTMs）of the identified cysteine site residues revealed that these sites undergo predominantly oxidative post-translational modifications.The results suggest that selenium probes have the potential to provide an ideal tool for probing proteins that regulate redox homeostasis and to facilitate the development of innovative selenium-based drugs.3.Using selenium probes combined with quantitative chemical proteomics strategies,we have systematically analyzed the dynamics of protein disulfide bonds in iron-dead cells and identified a series of candidate sites whose redox states are precisely regulated under iron-lethal induction and inhibition conditions.The discovery of these sites broadens the scope of new markers of iron death and is expected to accelerate the process of iron death research.4.Using the classical protein cysteine probe combined with quantitative chemical proteomics（rd TOP-ABPP）,we obtained its covalent modification sites in the proteome of two tumor cells.It was found that both high and low concentrations of ebselen covalently modified cysteine 53 on cell cycle protein-dependent kinase 5（CDK5）.In addition,high concentrations of ebselen resulted in changes in the expression of a variety of proteins involved in a wide range of functions and signaling pathways to the cell.5,A double click chemical probe capable of labeling both protein cysteines R-SNO and R-SOH was constructed using two types of bioorthogonal motifs.The bioorthogonal groups in the probes can be used for intracellular protein cysteine-SNO and-SOH imaging and chemical proteomic analysis,i.e.,as two oxidatively modified reactive groups and can be coupled to fluorophores or enrichment groups by click chemistry.Probes capable of simultaneously detecting two protein cysteine oxidative modifications have been synthesized and characterized for the first time and will provide an effective chemical tool for probing the dynamics of the two oxidative modifications under physiological and pathological conditions.