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毛细管筛分电泳中电动超荷电结合预填充水塞在线富集十二烷基磺酸钠-蛋白质的复合物(英文)

刘晶#, 康名超#, 刘震*   

  1. 生命分析化学国家重点实验室, 南京大学化学化工学院, 江苏 南京 210093
  • 收稿日期:2011-08-15 修回日期:2011-08-30 出版日期:2011-09-28 发布日期:2011-10-28
  • 通讯作者: 刘震，博士，教授，博士生导师.Tel/Fax: (025)83685639

On-line preconcentration of sodium dodecyl sulfate-protein complexes using electrokinetic supercharging method with a prefilled water plug in capillary sieving electrophoresis

LIU Jing#, KANG Mingchao#, LIU Zhen*   

  • Received:2011-08-15 Revised:2011-08-30 Online:2011-09-28 Published:2011-10-28
  • Contact: LIU Zhen, PhD, Professor. Tel/Fax: (025)83685639
  • Supported by:

    New Century Excellent Talents Program (No. NCET-08-0270) and the Fundamental Research Funds (No. 1093020506) from the Ministry of Education of China

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摘要：

提出了一种应用于毛细管筛分电泳中的电动超荷电结合柱头水塞堆积样品的方法,实现了十二烷基磺酸钠-蛋白质复合物的在线富集。一般情况下,电动超荷电方法是一种将电动进样与瞬时等速电泳联用的富集技术。具体过程是,首先在毛细管中注入背景电解质,再注入适量的前导电解质,然后电动进样一段时间。最后注入后导电解质开始瞬时等速电泳及分离的过程。本文在常规的电动超荷电技术基础上,在电动进样之前先注入一段含有聚合物的水塞以进一步提高富集效果。同时,考察了电动超荷电中不同富集方法叠加联用的效果,包括聚合物的筛分效应、结合水塞和不结合水塞的场放大样品进样效果、瞬时等速电泳等。结果表明,由于十二烷基磺酸钠-蛋白质复合物的质荷比接近,电动进样中的进样歧视得到消除,电动超荷电结合含聚合物水塞堆积样品的方法可以无歧视地在线富集十二烷基磺酸钠-蛋白质复合物,检测灵敏度增强1000倍以上。该方法非常适用于低丰度蛋白质的分析,并可同时提供相对分子质量信息。

关键词: 场放大进样, 蛋白质, 电动超荷电, 毛细管筛分电泳, 在线富集

Abstract:

An electrokinetic supercharging (EKS) method with a prefilled water plug at the head column of capillary was developed for on-line preconcentration of sodium dodecyl sulfate (SDS)-protein complexes in capillary sieving electrophoresis (CSE). Conventional EKS is a combination of electrokinetic injection with transient isotachophoresis (tr-ITP). The capillary is first filled with background electrolyte, then an appropriate amount of a leading electrolyte is filled and electro-injection is carried out for certain duration. After that, terminating electrolyte is filled, and tr-ITP is subsequently initiated, followed by capillary electrophoresis (CE) separation. In this work, the performance of EKS was evaluated by integrating multiple sub-methods step by step, and a water plug containing polymer was introduced before electrokinetic injection in order to further improve the concentration effect. The positive effects of the sub-methods were verified, including molecular sieving effect of polymer, field enhanced sample injection (FESI) with and without a water plug, and transient isotachophoretic electrophoresis-based FESI. It was observed that analyte discrimination usually encountered in conventional electrokinetic injection was eliminated due to the similar charge to mass ratios of SDS-protein complexes. Based on these results, a hybrid on-line preconcentration method, EKS with injecting a water plug containing polymer before sample electrokinetic injection, was proposed and used to indiscriminately preconcentrate SDS-protein complexes, which provided a sensitivity enhancement factor of more than1000. It was very suitable for the analysis of low-abundance proteins, providing the information of their molecular mass.

Key words: electrokinetic supercharging, on-line preconcentration, capillary sieving electrophoresis, field enhanced sample injection, protein