Chemical Labeling Of Protein Cysteine Residues And Its Application In Chemical Proteomics

Cysteine is one of the 20 natural amino acids that constitute proteins.Its side chain sulfhydryl group is easily stimulated by reactive oxygen species to undergo oxidation modification,and participates in redox regulation in organisms,which is closely related to many physiological and pathological processes.Due to the unique nucleophilicity of its side chain sulfhydryl group,the biological coupling reaction of cysteine has always been one of the hot topics of protein modification.However,most of the existing cysteine biological coupling reactions have some shortcomings such as poor selectivity and low reaction efficiency.In this work,based on the theory of soft,hard acid and base,and using the special hydrogen bond between pyridine and sulfhydryl,the cysteine labeling reagent was designed and optimized,and 2-pyridynone series compounds were synthesized without affecting the reaction rate and Under the premise of yield,the selectivity of the labeling reagent to cysteine is improved.We first studied the model reaction and found that 2-pyridynone derivatives reacted efficiently and quickly with cysteine substrates.By comparing the difference of the interaction between different groups and sulfhydryl groups,the transition state structure of the reaction of 2-pyridynone compounds with cysteine was predicted;combined with the model reaction data,the reaction mechanism was discussed.Subsequently,we further applied the obtained labeling reagent to the labeling of protein cysteine residues.The efficiency of labeling cysteine residues of recombinant proteins with this kind of labeling reagents was explored by Western blotting and fluorescent protein imaging.Finally,we used 2-pyridynone labeling reagents to label trastuzumab and explored its potential application value in antibody-drug coupling;we also successfully applied this reagent to the study of chemical proteomics,The labeling reaction of HeLa cell lysate can capture more than 4000 cysteine sites within 5 minutes,which is faster and more efficient than traditional iodoamide reagents.