Modified Of SiO₂ With Amino Acids/Dipeptide For Enrichment Of Glycopeptides

Protein glycosylation is one of the most studied post-translational modifications and glycoproteins play major roles in many biological processes.Moreover,many diseases are related to abnormal glycosylation of proteins.While glycoprotein analysis is technically challenging because of the glycan structure intricacy,linkage diversity,glycan heterogeneity and relatively low abundance.Many approaches for glycoprotein/glycopeptide enrichment have been developed such as lectin-based affinity chromatography,boronic acid chemistry,hydrazine chemical reaction,hydrophilic interaction liquid chromatography and so on,but they show some problems such as low coverage or low enrichment selectivity.It is urgent to develop a new method to separate and enrich the glycoproteins/glycopeptides.This study paid attention on this issue,and it was divided to 2 parts:In first part,inspired by the lectin-based affinity chromatography and hydrophilic interaction liquid chromatography,we selected amino acids as saccharide binding platform.Proline which is nitrogen-containing compounds and glutamic acid which is hydrophilic amino acid were grafted onto the surface of silica microspheres via atom transfer radical polymerization respectively.The two kinds of silica microspheres were characterized by SEM,TG and Nitrogen adsorption-desorption isotherms,and they exhibit good performance in glycopeptide enrichment,but could not working so well under the interference of nonglycopeptides.In second part,we synthesized Pro-Glu(PE)as the platform to bind saccharides by solid-phase peptide synthesis.First,we studied the binding properties between PE and seven monosaccharides which are very common in glycopeptides by fluorescent titration experiment,and calculated the association constants between PE and the monosaccharides.The values of the seven association constants ranged from hundreds to thousands,and it meaned that PE had different affinity to those monosaccharides.Then the silica microspheres onto which PE has been grafted,were used to test the properties of chromatopraphic separation for sialyated oligosaccharides with different linkage types.The results showed that our materials had good performance.Next,we studied the effect of our materials in glycopeptide enrichment,the results obtained from mass spectras showed that it could enrich glycopeptides so well even if the existence of 100 bolds of nonglycopeptides,which meaned our materials has anti-interference capacity,superior to commercial materials.In summary,the amino acids or oligopeptides can be used as saccharides platforms to design the materials for glycopeptide enrichment.This can help to develop more efficient and specific methods for the separation and enrichment of glycopeptide.