| Recombinant human erythropoietin is a glycoprotein with the major function of stimulation of erythropoiesis and applied to cure anemia due to kidney disease in clinic. Mass Spectrometry(MS) has been widely used in chemistry, biology, medical and pharmaceutical science field for its high sensitivity and selectivity. To build the effective method based on the mass spectrometry for the characterization of glycoprotein like EPO, we developed and optimized a series of methods to dectect the primary structure of glycoprotein, including molecular weight, content of sialic acid and carbohydrate etc., in rhEPOs by Mass Spectrometry(MS) with the sample of rhEPO concentrated solution. Based on the developed methods, four kinds of rhEPO coming from different manufactories were determined. The coverage of PMF of all rhEPOs reached 98% by optimization of digestion condition, and N-glycan site and two disulphide bridges in the rhEPOs were determined successfully. Moreover, the difference of the glycan structure type between these four rhEPOs was also probed. Our research work laid the foundations for developing rapid and sensitive detection method for primary structure determination of rhEPO by MS and provide valuable parameters for quality control of this drug.Wheat germ lectin(WGA) has specific affinity to N-acetylneuraminic acid and (β1-4 and β1-2 linked N-acetylglucosamine. It has been widely used for specific purification of glycoproteins from a protein mixture in proteomics research, which can simplify the complexity of the system and improve the efficiency of glycoprotein identification. The feasibility to selective separation rhEPO from protein mixture with WGA was tested in this research. The result shows that WGA can react strongly with the... |
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