Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as the standard proteomic approach for separating proteins in a complex mixture, this technique has many drawbacks. These include a limited molecular mass range, poor separation of highly acidic or basic proteins, and exclusion of the majority of membrane proteins from analysis. Considering the important functions of many membrane proteins, such as receptors, ion transporters, signal transducers, and cell adhesion proteins, it is increasingly important that these proteins are not excluded during the global proteomic analysis of cellular systems.MultidimensionalProteinIdentificationTechnology (MudPIT) offers a gel-free alternative to 2D-PAGE for the analysis of both membrane and soluble proteins. The goal of this chapter is to provide detailed methods for using MudPIT to profile both membrane and soluble proteins in complex unfractionated samples. Methods discussed will include tissue homogenization, sample preparation, MudPIT, data analysis, and an application for the analysis of unfractionated total tissue homogenate from human heart.
