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

University Of Delaware, Newark DE

Investigators

Abstract

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.