Liquid-based free-flow electrophoresis-reversed-phase HPLC: a proteomic tool

Moritz, RL; Simpson, RJ

Author(s): Moritz, RL; Simpson, RJ;
Details: Publication Year 2005-11,Volume 2,Issue #11,Page 863-873
Journal Title: NATURE METHODS
Publication Type: Journal Article
Abstract: One of the central problems of mass spectrometry (MS)-based proteomic analysis of complex protein mixtures is the issue of the wide dynamic range of protein abundances, which may vary from 10(5) to 10(6) for cells and log to 10(9) to 10(10) for tissues such as blood. To overcome this impasse, many prefractionation methods for simplifying complex protein mixtures, based on either electrophoretic, chromatographic or orthogonal electropheretic-chromatographic principles, have been developed(1,2). This protocol describes an orthogonal separation procedure utilizing a Liquid-based isoelectric focusing (IEF) protein separation method, free-flow electrophoresis(3) (FFE) in the first dimension and rapid off-line reversed-phase high-performance Liquid chromatography(4,5) (RP-HPLC) of each FFE pool in the second dimension. In the FFE step, complex protein mixtures are continuously injected into a carrier ampholyte solution flowing as a thin Laminar film (0.4-1.0 mm) between two parallel plates (Fig. 1). With the introduction of an electric field perpendicular to the direction of flow, proteins are separated by IEF according to their pI values and collected into 96 well-defined pools, each separated by similar to 0.02-0.10 pH unit. The nature of the pH gradient can be customized by the judicious choice of ampholytes(5). IEF can be performed in either nondenaturing or denaturing conditions by the addition of a chaotropic agent (for example, 6 M urea) and a reducing agent (for example, dithiothreitot (DTT)) in the separating buffer. Each FFE pool is then subjected to either analytical (for example, similar to 2.5% of FFE pool) or preparative rapid RP-HPLC (similar to 1-6 min per either analysis).
Publisher: NATURE PUBLISHING GROUP
Keywords: GEL-ELECTROPHORESIS; FLUORESCENT DYES; PH GRADIENTS; PROTEINS; LINE; SEPARATION; PEPTIDES; STRATEGY
Publisher's Version: https://doi.org/10.1038/nmeth1105-863
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2005-11-01 12:00:00