毛细管区带电泳用于血浆蛋白质混合物的分析

Capillary zone electrophoresis for plasma protein mixture analysis

目的探索建立采用毛细管区带电泳(CZE)法分析血浆相关蛋白质的条件和方法。方法 1)以血浆COHN法组分Ⅳ(CFⅣ)抽提液为样品,代表蛋白质混合物,分别从波长、基本缓冲液、添加剂、PH、温度等方面,摸索CEZ分析蛋白质混合物的较优条件和方法;2)将摸索得到的较优条件下的CZE分析CFⅣ抽提液的结果,并与通过高效液相色谱(HPLC)法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法及双向电泳(2-DE)法分析CFⅣ抽提液的结果比较;3)采用较优条件下的CZE法分析标准血浆、标准血浆经离子交换层析(IEC)的FⅧ粗提液等,考察该条件是否适用于血浆中的其他组分。结果 1)CZE法分析CFⅣ抽提液的较优条件为:20℃以214 nm紫外吸收波长,0.5 psi压力进样10 s,10 kV恒压分析50 min,缓冲体系为pH 9.0的0.04 mol/L硼酸硼砂、0.05%SDS、0.007 5%腐胺;2)采用此条件的CZE法分析CFⅣ得到14个蛋白峰、分析时间50 min,HPLC法分析得到10个蛋白峰、分析时间60 min,SDS-PAGE法分析得到11条蛋白条带、分析时间120 min,2-DE法分析得到14个蛋白点数(与CZE分析的蛋白峰数数量相同)、分析时间3 d;3)CZE法用于标准血浆、标准血浆经离子交换层析的FⅧ粗提液的分析可分别分辩出22和11个蛋白峰。结论以CZE法分析血浆相关蛋白混合物与现有分析方法比较,能达到较好的分析效果,经摸索确定的较优分析条件具有一定的通用性。

Objective To establish a universal capillary zone electrophoresis （CZE） condition for analysis of plasma protein mixtures. Methods 1 ） CFIV was chosen as the representative of plasma protein mixture to determine the best conditions for CZE by investigating wavelength, basic buffer components, buffer additives,pH and temperature. 2）We compared the results of CZE under the optimized condition with other conventional methods, such as high performance liquid chromatography（ HPLC ） ,dodecyl sulfate sodium salt polyacrylamide gel electrophoresis（SDS-PAGE） and two-dimension electrophoresis （2-DE）. 3 ）We used the optimized condition of CZE to analyze other plasma protein mixtures, such as standard plasma and coarse plasma factor Ⅷ （ FⅧ） mixture derived from ion exchange chromatography （IEC） to determine whether this condition was applicable to other plasma mixture samples. Results 1 ）The optimum conditions of CZE analysis of CFⅣ were as follows：absorption wavelength 214 nm,temperature 20℃ ,sample injected at 0. 5 psi pressure for 10 sec,10 kV for the 50 min＇ analysis process and the basic buffer component of 0. 04 mol/L boric acid borax buffer at pH 9. 0,containing 0. 05% （w/v） SDS,0. 007 5% （w/v） putrescine as buffer additives. 2）In CFⅣ analysis, using the optimized CZE condition, 14 protein peaks were obtained with a separation time of 50 min;10 protein peaks were given by HPLC, with a separation time of 60 min;11 protein bands were found by SDS-PAGE, and the separation time was 120 min; 2-DE gave 14 protein spots,which was the same number as the one obtained by CZE,but the separation time was 3 day. 3） The analysis of standard plasma and coarse human FVⅧ mixture by CZE under the optimized condition gave 22 and 11 peaks, respectively. Conclusion CZE method that we have established for analyzing plasma protein mixture could achieve a better result compared with the other three conventional methods.