HPLC and Mass Spectrometry of Intrinsic Membrane Proteins

Abstract

High-performance liquid chromatograpghy (HPLC) separations of membrane proteins can be conveniently divided into two categories. First, there are many methods available for isolation of functional membrane proteins. Typically, the proteins are maintained in configurations as close to their native state as possible through the use of mild detergents that provide solubility without denaturation, allowing convenient ion-exchange or size-exclusion chromatography, for example. Proteins or complexes isolated in this way are subsequently used for functional analysis or crystallization, and so on. These isolation techniques have been well reviewed and readers are referred to literature specific to the protein of interest. The second category of separations are those used to separate membrane proteins from detergents and salts for the purpose of protein chemistry; although tempting to call these methods “denaturing” there is substantial evidence that this is not always the case. The focus of this chapter is to review the latter category of HPLC techniques with specific reference to those methods that provide conditions compatible with mass spectrometric analysis, especially on-line electrospray ionization.