Glycoprotein Purification Using Magnetic Particle-Lectin Conjugates

Purpose: To establish a new lectin immobilization method supported by epoxy-coated magnetic particles. LCA bonding glycosylated proteins and sialic acid protein were fractioned and purified by this method from various biological samples, and were analyzed by 2D technology preliminarily.Method: We used a new lectin affinity method supported by functionalized magnetic particles to fractionate and enrich the glycosylated proteins. This method of using epoxy-coated magnetic particles coupled with LCA (Lens Culinaris Agglutinin) or WGA (Wheat Germ Agglutinin) to separate the glycosylation protein from total protein was established and optimized. We also study the effect ofα-methyl-D-mannoside and bivalence metal ion on LCA coupling to epoxy-coated magnetic particles. The elution buffer was selected and its concentration was determined. The anosia human liver cell extraction and liver cancer cell extraction was fractioned by this method respectively. Two-dimensional electrophoresis (2DE) was used for the separation of fractionated glycosylated proteins, and the 2D gels between anosia human liver cell (Chang Liver) and liver cancer cell (SMMC-7721) was compared with 2D gel analysis system.Result: The coupling rate between LCA and epoxy-coated magnetic particles could be up to 90% through optimization of coupling pH, temperature, time and confining concentrate. The effect ofα-methyl-D-mannoside and bivalence metal ion on LCA coupling to epoxy-coated magnetic particles is not significant. The 0.1% SDS could provide a suitable effect in the elution process. We isolate the LCA binding glycosylated proteins from anosia human liver cell extraction and liver cancer cell extraction by LCA coupled epoxy magnetic particles, and the glycosylated fractions were separated by 2D gels which were analyzed by 2D ImageMaster to established disparity expression spectra between Chang Liver and SMMC-7721 firstly. 148±5 and 209±7 spots were detected between 2D gels of Chang Liver and SMMC-7721 respectively and 127 spots were present in both samples but with varied expression levels, the match rate was 85.36%. 6 spots were up regulated and 3 were down regulated in the SMMC-7721 glycosylated proteins compared to Chang Liver, 3 spots absent and 35 new present. The reproducibility of proteins spots on two gels was regarded to be satisfactory in this experiment. The deviation of two gels in IGF direction is (0.873±0.215) mm, the deviation of two gels in SDS-PAGE direction is (1.025±0.125) mm. The WGA immobilization rate can reach 85% in acetous condition after 1h incubation. Good separating effect was provided through application in anosia human SDS-PAGE.Conclusions: Lectin immobilization method supported by epoxy-coated magnetic particles can be used as a tool to fraction the glycoproteins from complex samples and was used in study of glycoproteomics response in liver cancer, and a disparity expression spectra was established by this method. Different lectin can be coupled with epoxy-magnetic particles in different conditions to form various lectin affinity magnetic particles used for isolation of variform glycoprotein.