Research Of Phosphopeptide Enrichment Material: Based On Thioureidobenzoic Acid

The reversible phosphorylation of protein is one of the most common post-translational modifications in eukaryotic cells,and plays a very important role in regulating various biological cell processes.Substantial evidences have shown the close relationship between the abnormal protein phosphorylation and many critical diseases.Therefore,increasing attention has been paid to the research about phosphorylated proteins.However,the MS-based phosphoproteomics research of real biosamples is largely hampered by the interferential signals of non-modified proteins with relatively high abundance.And the highly dynamic nature and low stoichiometry of protein phosphorylation poses a great challenge to the enrichment of phosphorylated proteins/peptides and localization of phosphorylation sites.Over the past decade,the demand for in-depth phosphoproteomics studies has spawned a variety of selective affinity materials capable of capturing trace amounts of phosphorylated proteins/peptides from highly complex biosamples.But some problems still exist in enrichment capacity,sensitivity and selectivity.Aiming at this hotspot and difficulty in the field of the phosphopeptide enrichment,in this study a novel material was developed based on the interaction between thiourea receptors and phosphate group.3-thioureidobenzoic acid monolayer-based SiO₂ material was prepared to enrich phosphopeptides.First,the interaction between 3-thioureidobenzoic acid and phosphopeptides as well as its binding ability was investigated utilizing FT-IR,fluorescence titration,¹H NMR titration and QCM analysis.Then 3-thioureidobenzoic acid was grafted on the porous SiO₂ material using silane coupling agent as a medium.Finally,its ability to enrich phosphopeptides from standard phosphorylated protein digests was evaluated by mass spectrometry.TG,XPS and other characterization methods were used to confirm that the porous SiO₂ microspheres have been successfully modified by 3-thioureidobenzoic acid monolayer.At last the enrichment ability was assessed utilizing mass spectrometry combining with the results of QCM analysis.The results showed that the material we prepared can separate and enrich phosphopeptides in some extent,especially to multiply phosphopeptides.In view of all above analysis,the material developed based on the interaction between thiourea receptors and phosphate group could effectively separate and enrich phosphopeptides.This would provide some enlightenment for the development of novel phosphopeptides enrichment materials.