ABSTRACT

Progress in proteome analysis relies heavily on development of technologies to reduce the complexity of the sample prior to systematic application of such analytical techniques as gel electrophoresis, liquid chromatography, and mass spectrometry. Free fl ow electrophoresis (FFE), available from Becton, Dickinson and company (BD™), is reemerging as one of the most versatile semipreparative to preparative fractionation and separation techniques.1-4 It offers a unique and highly powerful approach to the separation of charged species including proteins, peptides, cellular organelles, and whole cells.5 Such a variety of FFE applications is achieved via different separation techniques: isoelectric focusing (IEF), zone electrophoresis (ZE), and isotachophoresis (ITP).6-9 This chapter focuses on the IEF mode of the FFE as it applies to analysis of proteins. Due to its matrix-free fractionation principle, FFE offers considerable

advantages over traditional chromatographic and gel-based techniques: exceptional sample recovery, fast fractionation times, and high throughput.10 These features allow FFE to offer a unique approach to reducing complexity of any proteome, often as a complementary technique to one-dimensional (1D) or two-dimensional (2D) gel electrophoresis.11 At the same time, continuous fl ow principle allows for virtually unlimited preparative fractionations.12 This combination of effective fractionation with high loading capacity provides for a practical methodology to enrich for low-abundant proteins and to segregate highly abundant proteins in biological samples such as cell lysates or body fl uids. This chapter is meant to give an overview of practical aspects of the free fl ow isoelectric focusing (FFE-IEF) and to demonstrate its capabilities using two challenging applications: display of low-abundant proteins in rat liver lysate and fractionation of human plasma. A detailed description of FFE protocols and a comprehensive review of literature and theoretical considerations have been recently published.13,14

The main functional part of an FFE device is a precisely manufactured chamber formed by two narrowly spaced plates (the distance between the plates is 0.4 or 0.5 mm), with the length of 500 mm and a width of 100 mm. Along the sides of the chamber, there are two electrodes that provide high voltage during separation. A sample is continuously infused into a thin, laminar layer of one or more separation buffers fl owing through the chamber. A voltage is applied perpendicularly to the fl ow direction. As separation buffer and samples are moving through the chamber, the electric fi eld leads to a defl ection of different sample components according to their charge. As a result, sample components that entered the chamber as a mixture at one end will leave the chamber at the other end as separated components that can be collected (Figure 11.1).