Topology of membrane proteins in native membranes using matrix-assisted laser desorption ionization/mass spectrometry

Abstract

Publisher Summary Information on topology is important for understanding the structural basis underlying the translocation function of cation pumps like the Na,K-ATPase, Ca-ATPase or H,K-ATPase. Previous efforts to define topology have used approaches like hydropathy plots, proteolysis of vesicles, binding of regio-specific antibodies, or labeling with group-specific, membrane-sided reagents followed by identifying the modified sites. Proteolysis of sided vesicles followed by analysis of peptide products has been one of the most common approaches to determine exposed peptide sequences. Conversely, remaining membrane-associated peptides can be analyzed after exhaustive protease digestion. In the study in this chapter, matrix-assisted laser desorption ionization/mass spectrometry (MALDI/MS) is used to identify the peptides released from gastric parietal cell microsomes. MALDI, because of its sensitivity and relative tolerance to the presence of salts and buffers is examined for the analysis of unfractionated proteolytic digests. MALDI with postsource decay (PSD) analysis has been used to obtain sequence information on peptides even in crude digestion mixtures. The strategy in this chapter consists of proteolysis of intact vesicles, centrifugation at high speeds to separate membrane bound and soluble fractions and analysis of the mixture of released peptides by MALDI/MS. In addition, to increase the sensitivity and breadth of analysis, supernatant peptides are separated by reverse-phase high-pressure liquid chromatography (HPLC) and individual fractions are analyzed by MALDI/MS. PSD-analysis is also performed to obtain partial sequence information and identify peptides. On the basis of the released peptide products, a topological map for a major portion of the H,K-ATPase in gastric parietal cell tubulovesicles is proposed. The chapter focuses on the gastric H,K-ATPase as a test protein because purified gastric microsomal vesicles are well-enriched in the enzyme, the vesicles are oriented with a common asymmetry—that is, cytoplasmic side out, the vesicles are sealed allowing selective cytoplasmic digestion, and there is a pool of existing topological data from other methods.