Protein Characterization by Capillary Isoelectric Focusing, Gas-Phase Electrophoretic Mobility and Mass Spectrometry

该奖项将支持开发一种新的仪器和相关方法，用于分离和表征蛋白质混合物中的单个组分。 该仪器由一个自动毛细管电泳（CE）系统耦合到气相离子迁移率分析仪（GEM）。 CE单元通过毛细管等电聚焦（CIEF）提供分子的初始分离;通过GEM分析仪对CIEF中出现的分子进行大小测定和计数。 该分析仪包括电荷还原电喷雾源、微分迁移率分析仪和凝聚粒子计数器。 电喷雾采用CIEF流出物并提供单电荷的单个分子用于气相分析。 由于气相离子迁移率谱可以快速获得，因此可以几乎与CIEF一样快速地获得完整的三维表征（分子数vs.气相迁移率vs.等电点）。开发工作将集中在两个主要领域：开发CIEF和GEM之间的有效接口，以及GEM输出与常规蛋白质质谱仪的有效耦合，蛋白质组学研究取决于仪器方法的持续开发和应用，以分离、鉴定和测量生物样品中的蛋白质量。 二维凝胶电泳是目前蛋白质组学研究的核心分析和制备技术，但通常是一个耗时的过程。 它具有难以控制和难以替代地使用的附加缺点。 虽然在这个项目中开发的仪器不太可能提供2-D凝胶所允许的分辨率，但它的速度，重现性和操作简单性可能使其成为许多应用中的首选仪器，其中不需要最高程度的分离，并且需要进一步表征分离产物。

This award will support the development of a new instrument and related methodology for separation and characterization of individual components in protein mixtures. The instrument consists of an automated capillary electrophoresis (CE) system coupled to a gas-phase ion mobility analyzer (GEM). The CE unit provides initial separation of molecules through capillary isoelectric focusing (CIEF); molecules emerging from CIEF are sized and counted by the GEM analyzer. The analyzer includes a charge reduction electrospray source, a differential mobility analyzer and condensation particle counter. The electrospray takes the CIEF effluent and provides singly-charged, individual molecules for gas phase analysis. Because a gas phase ion mobility spectrum can be acquired quickly, a complete three dimensional characterization (number of molecules vs. gas phase mobility vs. isoelectric point) can be obtained almost as rapidly as CIEF can be performed. Development efforts will focus on two main areas: development of an effective interface between CIEF and GEM, and the effective coupling of the GEM output to conventional protein mass spectrometers.Research in proteomics is dependent upon the continued development and application of instrumental methods to separate, identify and measure the amount of proteins in biological samples. Two dimensional gel (2D-gel) electrophoresis is currently the core analytical and preparative technology of proteomics research, but is typically a time-consuming procedure. It has the additional drawbacks of being difficult to control and difficult to use preparatively. Although the instrument to be developed in this project is unlikely to provide the resolution permitted by 2-D gels, it's speed, reproducibility and simplicity of operation are likely to make it the instrument of choice in a number of applications where the highest degree of separation is not necessary, and where further characterization of the products of separation is desired.