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DEVELOPMENT OF CAPILLARY AND MICROCHIP SEPARATION-DETECTION SYSTEMS TOWARD PROTEOMICS

面向蛋白质组学的毛细管和微芯片分离检测系统的开发

基本信息

批准号：
15350041
负责人：
OTSUKA Koji
金额：
$ 9.34万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Toward the high performance separation and detection of proteins by capillary electrophoresis(CE) and microchip electrophoresis(MCE), following five subjects were mainly investigated.1)Development of a high performance analysis method by microchip isoelectric focusing(MCIEF). Four model proteins were successfully separated within 210 s by MCIEF, where good linear relationship between a pI value of the protein and its focused position.2)Development of a highly efficient chiral separation technique by capillary electrochromatography and microchip electrochromatography using immobilized proteins. Avidin was used as a chiral selector and four pairs of enantiomers were successfully resolved.3)Improvement of detection sensitivity in MCE using on-line sample preconcentration. Under a micellar electrokinetic chromatography (MEKC) mode in MCE, successful on-line sample preconcentration by SRW was achieved by using a newly designed and fabricated T -cross type channel quartz chip.4)Improvement of detectability in CE and MCE by using thermal lens microscopy (TLM). In a microchip MEKC-TLM system with sweeping as an on-line sample preconcentration technique, the limit of detection of 4 nM or 8 amol in absolute amount injected for Sunset Yellow was obtained.5)Development of a high performance MCE system with mass spectrometric (MS) detection. An MCE-MS system with an electrospray ionization interface was developed, where some peptides and proteins were successfully separated and detected.

本文针对毛细管电泳（CE）和微芯片电泳（MCE）对蛋白质的高效分离和检测，主要开展了以下五个方面的研究：1）建立了一种高效的微芯片等电聚焦（MCIEF）分析方法。4种模型蛋白质在210 s内得到成功分离，其pI值与聚焦位置之间具有良好的线性关系。2）建立了固定化蛋白质的毛细管电色谱和微芯片电色谱高效手性分离技术。以亲和素为手性选择剂，成功拆分了4对映体。3）在线样品预富集提高了毛细管电泳检测灵敏度。在胶束电动色谱（MEKC）模式下，采用自行设计和制作的T -十字型通道石英芯片，成功实现了SRW在线样品预富集。4）采用热透镜显微镜（TLM）提高了毛细管电泳和胶束电动色谱的检测灵敏度。在采用扫描作为在线样品预浓缩技术的微芯片MEKC-TLM系统中，日落黄的绝对进样量检测限为4 nM或8阿莫尔。5）开发具有质谱（MS）检测的高性能MCE系统。建立了一种具有电喷雾电离接口的MCE-MS系统，成功地分离和检测了一些多肽和蛋白质。