Synthesis Of Glcnac Functionalized Magnetic Nanomaterials For Enrichment Of Glycopeptides

Protein glycosylation is one of the most important and common modifications after translation.It plays an important role in regulating cell-cell interaction,molecular recognition,protein folding and other complex biological processes.Many human disease is related to abnormal N-glycosylation.At present,mass spectrometry has become the primary technology to characterize protein glycosylation.However,the inherent low abundance of glycopeptides,the heterogeneity of glycosylation sites and the serious ion inhibition caused by the coexistence of non glycosylated peptides make the direct mass spectrometry analysis of glycopeptides still a challenge.Therefore,an effective enrichment process is needed before mass spectrometry.In the fields of biological molecules,targeted drugs,cancer treatment and other biotechnology,magnetic nanomaterials have broad application prospects.In this paper,functional magnetic nanoparticles with good dispersion,biocompatibility,non-toxicity and strong external magnetic response were prepared by hydrothermal method,which was applied to the enrichment of glycopeptides in trypsin digestion products.The work is as follows:The functional magnetic nano material Fe3O4@NH2 was prepared by hydrothermal method;Two methods were designed to prepare the functional magnetic material Fe3O4@NH2-GlcNAc.The first method was that DCC is used as a condensation agent to activate the carboxyl group of Boc-Asn-GlcNAc,then combined with Fe3O4@NH2 amino group to form the amide bond to obtain the protein coated magnetic composite particles with oligosaccharide;The second method was that polyethyleneimine combined with Boc-Asn-GlcNAc.The polymer was prepared by hydrothermal method with FeCl3·6H2O as iron source.Through the comparison of methods,the first method was selected for subsequent enrichment study,and the functional magnetic material Fe3O4@GlcNAe was prepared by hydrothermal method.2.The three functional magnetic materials were characterized by transmission electron microscopy(TEM),Fourier transform infrared(FT-IR)and X-ray diffraction(XRD).The results show that Fe3O4@NH2 has amino hydrophilic group,spherical shape,particle size of about 282nm;Fe3O4@NH2-GlcNAc has oligosaccharide hydrophilic group,low grafting rate.Fe3O4@GlcNAc has a monosaccharide hydrophilic group with a particle size of about 190nm.3.Combined with the advantages of hydrophilicity and magnetic separation technology,taking sialic acid glycopeptide(SGP)as the glycopeptide model,the application of glycopeptide enrichment was studied.The results show that Fe3O4@NH2-GlcNAc has a higher enrichment capacity than Fe3O4@NH2.Taking the mixture of glycopeptide and peptide as the research object,Fe3O4@NH2-GlcNAc has a little enrichment of hydrophilic group peptide,which is selective for hydrophilic macromolecules.The model of glycoprotein was studied with fetal globulin,the results showed that Fe3O4@NH2-GlcNAc was more selective than acetone.