The Hydroxyl-functionalized Magnetic Particles For Purification Of Glycan-binding Proteins

Background: Glycan-binding proteins (GBPs) could regulate many fundamental and important biological functions, including cell recognition, cell signaling pathway, growth, endocytosis, intracellular, apoptosis, cellular differentiation and infection of pathogenic microorganism through the interaction with glycan of glycoprotein or glucolipid. At present, the separation and extraction of GBPs are mainly through the mixed way of affinity chromatograph and ion exchange chromatography in abroad. However, this mixed method is not fit for the research on low abundance protein for its disadvantages, including nonidio-sticking, much time and resource consuming, complicated manipulation and the condensing need of interest protein. We established a new method for separation and extraction of GBPs basing on magnetic particles, which has many advantages, including high efficiency, convenience and exactness for separation and extraction of GBPs, taking the deficiency of the old ways into consideration.Purpose: To establish a new glycan immobilization method supported by hydroxyl-functionalized magnetic particles. Glycan-binding proteins were fractioned and purified by this method from various biological samples, and were analyzed by 2D technology preliminarily.Method: A rapid simple method for the purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate(mannose,glucose and cellobiose). All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from healthy human sera, Kashin-Bek syndrome sera and liver cancer human sera respectively. Two-dimensional electrophoresis (2DE) was used for the separation of fractionated glycan-binding proteins, and the 2D gels between healthy human sera and illness sera were analysed with 2D gel analysis system.Result: The results showed that the ability of coupling with glycan bonds by hydroxyl-functionalized magnetic particles was better than the other three magnetic particles through the comparing of the reduction of Fe₃O₄ magnetic particles, epoxy-coated magnetic particles, hydrazino-coated magnetic particles and hydroxyl-functionalized magnetic particles. Then the conditions of modification of the hydroxylation for magnetic particles were optimized, including the optimal modifying time, the amount of 4-hydroxybenzhydrazide and the temperature. The coupling amount could attain the maximum by optimizing the coupling buffer system, coupling pH and temperature. It was found that neither buffer system nor elution system had significant effect on conjugates through the research on their influence on the stability of magnetic particle-carbohydrate conjugates. The 0.1% SDS was confirmed to be the elution system for GBPs. The separation and purification of GBPs were carried out from healthy human sera, Kashin-Bek syndrome sera and liver cancer human sera, the identity and difference were also discovered by SDS-PAGE and silver staining. In the end, the diversity atlas on GBPs between healthy human sera and Kashin-Bek syndrome sera, and diversity atlas on GBPs between healthy human sera and liver cancer human sera were established respectively through the way of 2D electrophoretic technique and its image analysis.Conclusions: All kinds of reducing glycan could be immobilization modified when the hydroxylation-functionalized magnetic paricles were made as carriers.Under the same condition, many kinds of reducing glycan could be coupled efficiently. The method that separate and extract GBPs in the way of immobilizating reducing glycan chains with hydroxyl-functionalized magnetic particles can simply the protein ingredients, it could be made used in the research on proteome, too. The diversity expression atlas were established for GBPs of health human serum and illness serum, making use of 2D electrophoretic technique.