Identification of components of trans-Golgi network-derived transport vesicles and detergent-insoluble complexes by nanoelectrospray tandem mass spectrometry.

Epithelial cells have to deliver newly synthesized proteins to the apical and the basolateral plasma membrane domains of the polarized cell surface. Sorting takes place in the trans-Golgi network and at least two vesicular carriers exist for apical and basolateral delivery. After immuno-isolation, the composition of these vesicle preparations was analyzed by two-dimensional (2-D) gel electrophoresis and detergent extraction. In this paper we compare the constituents of detergent-insoluble complexes in different cell lines of polarized or nonpolarized origin and present the identification of five previously uncharacterized proteins. We show that our protein identification strategy can be successfully applied to the problem of small hydrophobic proteins from organisms that have not been substantially sequenced. The high sensitivity of nanoelectrospray tandem mass spectrometry allowed us to identify two proteins that belong to the p23/p24 family of putative cargo receptors for vesicular trafficking. Furthermore we have mapped CD9 and CD81, two members of a large family of proteins consisting of highly hydrophobic four transmembrane proteins. In addition we have identified caveolin-2 as a constituent of basolateral transport vesicles. We have also extended our analysis of immuno-isolated vesicles to a more basic pI range and show that this region on 2-D gels is devoid of proteins. With these approaches and with the previously published data we have now identified most of the major low molecular weight proteins recovered in detergent-insoluble glycolipid-enriched complexes.