Construction And Application Of Sulfhydryl Oligopeptide Enrichment System Based On PH Response

In proteome analysis,how to achieve efficient and specific enrichment of thiol peptides has always been a challenge.In response to this problem,firstly,based on magnetic nanoparticles,magnetic nanoparticles@5MP,a p H responsive sulfhydryl peptide trapping enrichment material,was prepared by the reaction of the exposed amino group with the 5MP.GSH was taken as a target to verify the enrichment,and the capture and release of GSH were explored.We took the chitosan derivative enrichment material as our experimental object.The effect of chitosan molecular weight on the stability of the grafted 5MP group under simulated p H response conditions was first explored,and then the thiol peptide GC-7 was used as the capture object to explore the effect of chitosan molecular weight on the stability of the grafted5MP group.The conditions for capturing/releasing sulfhydryl peptides by the material,and the8-peptide with high abundance and no sulfhydryl group was used as the interference target to verify the capture specificity of O-CMC-PBS-5MP for sulfhydryl-containing peptides;Finally,the feasibility of applying specifically enriched sulfhydryl groups peptides directly to LC-MS and MALDI-MS analysis was explored.The specific experiment content is as follows:（1）Two kinds of magnetic nanoparticles grafted with 5MP（Si O₂-NH₂@5MP and Si O₂-PEG-NH₂@5MP）and a derivative of chitosan grafted with 5MP group O-CMC-PBS-5MP were synthesized,the differences of GSH capture and release of thiol oligopeptide were compared.The capture conditions of magnetic nanoparticle materials are that magnetic nanoparticles@5MP and GSH oscillate in water bath at 37℃for 6 h under neutral conditions,and Si O₂-NH₂@5MP and Si O₂-PEG-NH₂@5MP.The capture rate of GSH by the two enrichment materials is about 30%,the release condition of Si O₂-PEG-NH₂@5MP@GSH and Si O₂-NH₂@5MP@GSH was 45℃water bath reaction 48 h at p H 11.0,and the release rates were 10%and 0%,respectively.The capture condition of O-CMC-PBS-5MP for GSH is that O-CMC-PBS-5MP and sulfhydryl oligopeptide GSH were bathed in water at 37℃for 12 h,the capture rate of sulfhydryl oligopeptide GSH can reach more than 90%,and the release condition is O-CMC-PBS-5MP@GSH when the p H is 11.0 and the reaction is carried out in 45℃water bath for 24 h,the release rate is about 54%,and the enrichment rate of GSH could achieve as high as 50%.（2）The influence of the molecular weight of chitosan on the stability of 5MP on the O-CMC-PBS-5MP was investigated.The 5MP group on O-CMC-PBS-5MP is the key group for capturing thiol peptides.The stability of grafted 5MP is extremely important.Therefore,chitosan with different molecular weights is used as raw material to synthesize the enriched material O-CMC-PBS-5MP with corresponding molecular weights.The results showed that the grafted 5MP on O-CMC-PBS-5MP synthesized with 400 KDa chitosan as the main chain is the most stable and most suitable for the enrichment of sulfhydryl peptides.O-CMC-PBS-5MP synthesized from KDa chitosan was used for the enrichment of thiol peptides.（3）The capture and release of sulfhydryl peptides by the enrichment material O-CMC-PBS-5MP were explored.The commercially purchased sulfhydryl peptide segment GC-7 was used as the enrichment object,and the capture condition was neutral,O-CMC-PBS-5MP and sulfhydryl peptide GC-7 were water bathed at 37°C for 12 h,the capture rate of sulfhydryl peptide GC-7 could reach 90%,the release rate could reach about 54%,and the specific enrichment rate of sulfhydryl peptide GC-7 could reach 50%.The enriched products can be directly applied to LC-MS and MALDI-MS analysis.