Proteomic characterization of membrane protein topology

Integral membrane proteins are represented by 20-30% of the eukaryotic genome and crucial for cellular functions including cell signaling, nutrient influx, toxin efflux, and maintenance of osmotic balance. Importantly, over 70% of all drugs are targeted at membrane proteins. Because of their hydrophobicity, however, methods used to characterize the structure of soluble proteins, such as NMR and X-ray crystallography, are generally not suitable to the study of membrane proteins (1). The methods described in this chapter facilitate the identification and mapping of both extracellular and cytoplasmicsoluble domains of integral plasma membrane proteins using mass spectrometry. By combining a classical protease protection approach with recently developed proteomic methods, protease-accessible peptides (PAPs) are digested from proteins embedded in their native lipid environment and identified to characterize the topologies of integral membrane proteins.