Preparation Of Poly-dipeptide Based Chromatographic Materials And Its Application In Erichment Of Glycopeptides

Protein glycosylation is one of the most important modifications in more than200 post-translational modifications,which plays crucial roles in the regulation of protein functions and numerous cellular processes,including cell adhesion,receptor activation,signal transduction,molecular transport and so on.It has been proved that abnormal glycosylation would change the structure and function of proteins,and even lead to the occurrence of cancers and other diseases.The study of glycoprotein is of great significance in studying its biological function and elucidating the mechanism of related diseases.In addition,identification of glycosylation sites and analysis of glycan structures are important components in current glycosylation studies.However,in complex protein samples,the interference of nonglycopeptides could strongly suppress mass spectrometry?MS?signals of low abundance glycopeptides.Besides,high degree heterogeneity of glycosylation also increased the difficulty of MS detection.Therefore,effective enrichment methods of glycoproteins/glycopeptides are very important to the study of glycoproteomics.However,current enrichment methods have numerous limitations,novel glycopeptide enrichment materials are needed to enrich low-abundance glycopeptides from complex biosamples.As a typical example,lectins could bind to specific saccharides via multiple hydrogen bonds.Inspired by this,a series of dipeptides were selected and two of them have been developed into novel glycopeptide enriched materials.In this experiment,the binding constants of 20 dipeptides with 7 monosaccharides were determined by fluorescence titration experiment.The dipeptide sequences were selected by the hydrophilic index in range of-3 to 3.Then,based on the combination of dipeptide hydrophilic index,dipeptide sequences of Pro-Asp?PD?and Tyr-Asp?YD?have been screened out,which can distinguish different monosaccharides.Then dipeptide functional monomers were prepared by grafting double bonds to the ends of PD and YD.After that,we synthesized homopolymers from functional monomers,which were coated on mesoporous silica gels by surface initiation-atom transfer radical polymerization.Then we prepared two kinds of dipeptide polymer modified chromatographicseparationmaterials.Inaddition,SiO₂@poly-PDand SiO₂@poly-YD were characterized by BET,TG,XPS and SEM.Through these characterization data,it has been proved that these polymers have been successfully grafted onto the surface of porous silica.The chromatographic separation experiments indicated that the liquid chromatographic column packed with these dipeptide polymers showed excellent separation ability for oligosaccharides and sialic acid derivatives.At the same time,by combining the polymer-modified silica materials with dispersive solid phase extraction mode,we found that these materials display excellent enrichment performance,SiO₂@poly-YD have anti-interference capacity capable of resisting100-fold bovine serum albumin interference.SiO₂@poly-PD have strong anti-interference capacity capable of resisting 200-fold bovine serum albumin interference,which has showed high anti-interference capacity in glycopeptide enrichment.By optimizing enrichment conditions,we believed that these kinds of smart polymer materials have potential applications in large scale qualification glycopeptides in complex human tissue samples,and will benefit future development of glycoproteomics.