Preparation Of Affinity Chromatographic Materials For The Separation And Analysis Of Glycoproteins/Glycopeptides

Glycosylation is one of the most common and important protein modifications, and more than 50% of all proteins in mammalian are estimated to be glycosylated. Glycosylation plays a crucial role in various biological functions, including cell growth, developmental biology, intracellular transport, immune response, cell to cell communications, protein folding, as well as cell and host-microbial recognition. Aberrant glycosylation is reported to associate with diseases such as cancers, human immunodeficiency virus, and immune disorders. It is considerably important to study protein glycosylation and the associated glycans for diagnostics, disease prognostics and pathological processes. Therefore it is absolutely necessary to develop efficient separation materials or separation methods for further analysis of glycoproteins or glycopeptides.In the present thesis, we dedicated to developing simple, efficient and economical method for preparation of silica-based stationary phase as well as the corresponding chromatographic method for the separation of glycoprpteins/glycopeptides.There are some limitations for traditional agarose-based affinity materials, such as unresistant to high pressure and low binding capacities. To solve the above problems, we developed a simple and easy accessible method to prepare silica-based material terminated with carboxyl groups. And lectin concanavalin A (Con A) was successfully immobilized onto silica materials via EDC/NHS activation to obtain the SG Con A material. Under high performance lectin affinity chromatography mode, the SG Con A material was successfully employed to selectively enrich glycoprotein ovalbumin with hybrid-type glycans and glycoprotein of RNase B with high-mannose type glycans compared to that of the commercial Agarose Con A material. The high efficiency of the SG Con A material demonstrated that it was promising in glycoproteomics analysis.Traditionally, the synthetic techniques in preparation of lectin material, were associated with non-specific adsorption could not be entirely eliminated during the affinity separations. Herein, using oxidized dextran as the spacer, a silica-based concanavalin A material (SiO2-ODex Con A) was synthesized to enrich glycoproteins/glycopeptides. For comparison, the SiO2-Ald Con A material was synthesized using conventional (3-glycidoxypropyl) triethoxysilane (GPMS) as the initial spacer arm. Taking advantage of the excellent features of oxidized dextran, low non-specific adsorption and high binding capacity of ovalbumin were achieved on the SiO2-ODex Con A material compared to the SiO2-Ald Con A material. Under high performance liquid affinity chromatography (HPLAC) mode, the SiO2-ODex Con A material was highly effective in the enrichment of glycoproteins/glycopeptides attached to high-mannose-type and bi-antennary complex-type glycans. The promising potential of the SiO2-ODex Con A material was demonstrated by fractionation of glycoproteins from complex biological samples for glycosylation analysis.Driven by the current growing demand for antibody therapeutics, it needs developing efficient chromatographic technology for downstream processing of antibody. Herein, the conjugation of divinyl sulfone (DVS) and 3-aminopropylsilane allowed to form a thiophilic anion exchange template to prepare functional stationary phases for antibody recovery. Facilitated by DVS chemistry, a silica-based material terminated with mannose and (OEG)4NH2 groups (SiO2-DVSM) was prepared conveniently. The material was characterized by elemental analysis, FT-IR. Zeta potential determination showed the sensitivity of the material to pH. IgG-binding capacity of the material was measured in different pHs covering a range from 4.20 to 8.00. The SiO2-DVSM material displayed high hydrophilic separation performance for mono-/oligosaccharide and anion exchange capability for nucleosides. By changing pH, a facile mixed-mode chromatograph has been developed for efficient production of IgG. Specifically, four fractions of IgG were eluted orderly by water, acid water (pH 3.0), and sodium acetate (10 mM, pH 3.2), and the sequence of each fraction was characterized by MS method. The promising potential of the SiO2-DVSM material was demonstrated by efficient fractionation of IgG from normal human serum, providing an accessible alternative method for antibody production.Taking advantage of the effectiveness and fast rate of the reaction, "click chemistry" has been widely used as an efficient template in preparation of high performance liquid chromatographic stationary phase. Unfortunately, the use of copper (I) in click reaction would cause toxic results for ligands or biomolecules to be separated, such as lectin, cell or protein, therefore, it is necessary to develop materials with copper free reaction for affinity chromatography. In the present study, the synthons of strain promoted azide-alkyne click reaction (SPAAC) were successfully prepared and applied to immobilize protein A molecules onto silica gels to obtain the SiO2-SPAAC Protein A material. Under HPLC mode, the SiO2-SPAAC Protein A material was specifically applied to enrich IgG from human serum sample, which were identified by MS analysis. The provided SPAAC reaction promised an accessible alternative method for preparation of affinity materials.