High Branched Polymer For Highly Efficient Enrichment Of Glycopeptide

As one of the most common post-translational modification, protein glycosylation not only plays key roles in the stability, solubility, folding and function execution of proteins, but also is involved in transportation, localization and recognition of protein in vivo.Furthermore, it has proved that abnormal glycosylation are associated with diseases and cancer, and many glycoproteins are also clinical biomarkers and therapeutic targets. Thus, the separation, detection and identification of glycoproteins/glycopeptides are of great importance, and it can help us to understand biological processes better, identify more potential diagnostic markers and therapeutic targets. Currently, glycopeptide qualitative and quantitative based on biological spectrometry mass analysis, has become an important field of researches on protein post-translational modifications. Since glycopeptides usually exist in very low abundance (2%～5%) in protein digestion, they are easily interfered by the highly abundanz non-glycopeptide during mass spectrometric analysis. In addition, the microheterogeneity of carbohydrate chain may further reduce the relative amount of glycopeptides and makes them rather difficult to be detected. Therefore, the selective enrichment of glycoproteins is of great importance. Lectins and antibodies are two major molecular tools for selective enrichment of glycoprotein or glycopeptides, but these biomolecules are generally expensive and have poor stability. Molecular exclusion,hydrophilic chromatography,boric acid concentration are also widely used in the enrichment of glycopeptides. However, these methods are all associated with apparent disadvantages, which making progresses in glycosylation research is relatively delayed than other post-translational modifications like phosphorylation, ubiquitination and acetylation. To date, the hydrazide chemistry based enrichment approach is one of the most frequently used techniques to isolate glycopeptides. But it also has some inevitable drawbacks:sample loss, complicated steps. Moreover, the most common commercial hydrazide resin could only provide limited binding affinity towards glycoproteins/glycopeptides.Therefore, it is essential to develop novel hydrazide-functionalized materials to improved binding strength of the reagent.High branched polymer are branched, three dimensional spherical with large number of end groups, which shows different properties from linear molecule,and shows promises in several biomedical applications. In recent years it become a hot research field of polymer science,including some of the applications in proteomics research.In the first chapter of this dissertation the progresses in the enrichment of glycoproteins/glycopeptides are reviewed. In the second chapter experiments were performed to verify that the polyamido-amine (PAMAM) dendrimer can be used for glycopeptide enrichment. The high density amine group bearing PAMAM with different generation (PAMAM-G4, PAMAM-G6) were linked to CNBr-activated Sepharose bead, and then used for isolation of glycopeptides from complex matrix. The method was optimized first with model glycoprotein, including reducing agents, binding solution, washing buffer and PAMAM to peptide ratio.In Chapter three, we synthesized a hyperbranched glycidol (HPG), and using SEM,’H-NMR, IR and other means to characterize the structure and external morphology. After the successfully synthesis of HPG, the polymer was fixed to the amino-silica micro particles, followed with adipic acid dihydrazide modification. A variety of methods were used to verify the new material and the results showed that the hydrazine reagent was successfully synthesized. We evaluated the application of the two novel reagents in the enrichment of mouse brain glycopepitdes, and both of them showed a high efficiency:with reagent based on PAMAM,204non-redundant glycopeptides from133glycoproteins were identified; with reagent based on HPG726non-redundant glycopeptides from308glycoproteins were identified. Our results showed that the high branched polymer material can be effectively used for the low-abundance glycoproteins/glycopeptides enrichment.