Capillary HPLC/electrospray ionization mass spectrometry for structural analysis of micro-quantities of proteins

毛细管 HPLC/电喷雾电离质谱法用于微量蛋白质的结构分析

基本信息

批准号：
06680584
负责人：
TAKAO Toshifumi
金额：
$ 1.28万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Capillary high-performance liquid chromatography (HPLC) has been combined with a magnetic sector mass spectrometer by improving the electrospray ion source. It allows the sensitive and accurate mass measurement in the HPLC/ESI-MS.As a result, all the components involved in a mixture sample such as an enzymatic digest of a protein could be efficiently measured following separation by a capillary column (300 mm diameter). Analyzes of a protein mixture and proteolytic digest indicate that the combination of a capillary HPLC and magnetic sector mass spectrometer is capable of attaining better than 0.01 % accuracy in mass determination on picomole quantities. It could successfully be applied to determination of the reading frames of DNA-derived protein sequence and both sites and types of modifications present in recombinant or natural proteins. A micro-blotting apparatus, which had been designed to be coupled with a capillary HPLC for collecting small volumes of sample solution eluted from a capillary column onto a polyvinylidene fluoride membrane, was demonstrated to fractionate each component separated by a capillary column with the high recovery (more than 90 %), which could readily be subjected to amino acid composition or sequence analyzes. The combination of a capillary HPLC/ESI-MS and micro-blotting system would go a good way for micro-analysis of peptides and proteins that are available only in limited quantities.

毛细管高性能液相色谱（HPLC）已通过改善电喷雾离子源与磁性扇形质谱仪结合使用。它允许在HPLC/ESI-MS中进行敏感，准确的质量测量，结果，在通过毛细管柱分离后，可以有效地测量混合物样品中涉及的所有组件，例如蛋白质的酶消化剂（300 mm直径）。对蛋白质混合物和蛋白水解摘要的分析表明，毛细血管HPLC和磁性扇形质谱仪的组合能够在picomole量的质量测定中获得高于0.01％的精度。它可以成功地应用于确定DNA衍生蛋白序列的阅读框，以及重组或天然蛋白质中存在的位点和修饰的类型。一个微吸收的设备，该设备被设计为与毛细管HPLC耦合，用于收集从毛细管柱上洗脱至聚偏二氟化物膜膜上的小体积样品溶液，可将每个组件分割为每个组件，由毛细管柱分隔，以高恢复（超过90％）的分析（可及时分析）。毛细血管HPLC/ESI-MS和微观印刷系统的组合将是仅以有限量可用的肽和蛋白质微分析的好方法。