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脉冲毛细管电泳分离蛋白质的研究
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作者单位
李振庆,李珊妮,朱月萍,张大伟,山口佳则 1. 上海理工大学 光电信息与计算机工程学院教育部医用光学仪器与设备重点实验室上海 2000932. 大阪大学先进光子研究中心大阪 565-0871 
基金项目:上海市2018年度“科技创新行动计划”(18441900400)
中文摘要:蛋白质是机体细胞的重要组成成分,通过分析蛋白质可获得机体的受损或病变情况,因此毛细管电泳(CE)分析蛋白质分子在临床医学中具有重要的应用价值。该文以溶菌酶、生长激素、碳酸酐酶、肌动蛋白、牛血清白蛋白和磷酸化酶B为样本,采用有效长度为6 cm和总长度为8 cm的毛细管,以羟乙基纤维素(HEC)为分离介质,首次采用脉冲电场毛细管电泳(PFCE),研究了脉冲频率及调制深度对不同蛋白质(分子量14.4~97.4 kDa)分离效率的影响。结果表明,相对于电场强度为100 V/cm的直流电场毛细管电泳(DCCE),在1.4% HEC筛分介质中采用平均电场强度为100 V/cm,脉冲频率为10 Hz,调制深度为250%的脉冲电场时,相同蛋白质分子的分离时间缩短6.6%~9.6%;脉冲电场与蛋白质分子的共振频率为10 Hz,当脉冲频率低于共振频率时,分离时间随脉冲频率的增高而延长,反之则随脉冲频率的增高而减少;当脉冲频率与共振频率相同时,其分离度提高34.1%~88.1%,理论塔板数最高为4.03 × 104;在相同平均电场下,当调制深度由0提至250%时,筛分介质内的焦耳热比直流电场时提高了2.64倍;焦耳热的提高使得筛分介质的粘度降低,导致蛋白质分子在筛分介质内的迁移时间随电场脉冲调制深度的增加而减少,且蛋白质的分子量越大,其迁移时间越小,其中磷酸化酶B的迁移时间减少约9.6%,但相邻蛋白质分子间的分离度无明显降低。该研究为提高CE对蛋白质分子的分离效率提供了新方法。
中文关键词:毛细管电泳(CE)  脉冲电场  蛋白质  分离
 
Study on Separation of Proteins by Pulsed Field Capillary Electrophoresis
Abstract:Protein is an essential component of organisms. Damages and diseases of the body could be known from analysis of proper proteins, and thus analysis of proteins by capillary electrophoresis(CE) is important in the field of clinical medicine. However, the current researches on pulsed field capillary electrophoresis(PFCE) mainly focus on the improvement of separation performance of DNA, and there is little research about protein analysis by PFCE. To improve the separation performance of proteins, lysozyme, growth hormone, carbonic anhydrase, actin, bovine serum albumin and phosphorylase B were taken as the samples, using hydroxyethyl cellulose(HEC) as the sieving polymer, applying the capillary with 6 cm of effective length and 8 cm of total length, performing the PFCE of proteins, and investigating the effect of pulse frequencies and modulation depth on the separation performance of proteins sized range of 14.4-97.4 kDa for the first time. Results demonstrated that, compared with the direct current CE(DCCE) under 100 V/cm of electric field strength, the PFCE employing 1.4% HEC as the sieving polymer, a 100 V/cm of average electric field strength, a 10 Hz of pulsed frequency and a 250% of modulation depth would make the migration time of proteins with same molecular weight decrease about 6.6%-9.6%. The resonant frequency for the protein and pulsed electric field was 10 Hz. The migration time of protein molecule was increased with the growth of pulsed frequency when the pulsed frequency was lower than the resonant frequency. In contrast, the migration time was decreased if the pulsed frequency was higher than the resonant frequency. The resolution could be increased about 34.1%-88.1%, and the maximum theoretical plate number reached to 4.03 × 104 if the frequency of the pulsed electric field was the same as the resonant frequency. Under the same average electric field strength, the migration time of protein decreased with the growth of modulation depth from 0 to 250%, and the Joule heating in the sieving polymer induced by pulsed electric field increased 2.64 times than that under direct current electric field. The growth of Joule heating will make the viscosity of the polymer reducing. Consequently, the decrease of the migration time was more obvious if the molecular weight of protein was larger. The migration time of phosphorylase B was reduced about 9.6% when the modulation depth was raised from 0 to 250%. The resolution between the adjacent protein molecules was not obviously deteriorated, even though the migration time was shorter. This work provides a new method for improving the separation performance of protein.
Key Words:capillary electrophoresis(CE)  pulsed electric field  protein  separation
引用本文:李振庆,李珊妮,朱月萍,张大伟,山口佳则.脉冲毛细管电泳分离蛋白质的研究[J].分析测试学报,2021,40(9):1361-1366.
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