Establishment and Application of ICIEF for the Analysis of Charge Heterogeneity of Human Urinary Kini-nogenase
OBJECTIVE To establish an analysis method of the charge heterogeneity of human urinary kininogenase(HUK)by using image capillary isoelectric focusing(iCIEF)and complete the methodological verification.METHODS The 35 μL 1％methyl cellulose,10 μL 200 mmol·L-1 IDA,4 μL pharmalyte electrolyte,48 mg urea,0.5 μL pI marker with pl 6.15 and 7.05 were added in the sample solution.The focusing condition was pre-focusing voltage 1 500V,duration 1 min,focusing voltage 3 000 V,duration 6 min.RESULTS The optimized method has stable baseline,and the target protein was significantly different from the unrelated protein,The recovery rate of accuracy verification was within 90％-110％,and the linearity verification result had r2 of 0.995 7,The RSD of each isomer pI in the repeatability verification was less than 0.2％.The limit of quantification was 0.013 mg·mL-1.The concentration of urea durability,IDA durability and the electrolyte pharmalyte durability are good.Using this method,the charge isomers of HUK from different manufactures were analyzed effectively.CONCLUSION The developed iCIEF method has good specificity,precision,linearity and durability,and can solve the problem of charge heterogeneity evaluation of protein products,which is of great significance to the quality control of such products from the perspective of charge heterogeneity.
human urinary kininogenaseglycoproteincharge heterogeneityimage capillary isoelectric focusing(iCIEF)
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