Proteomic Analysis of Surface and Endosomal Membrane Proteins from the Avian LMH Epithelial Cell Line

Proteins at the cell surface and within the endocytic pathway are increasingly being recognized for their roles in a wide variety of intercellular interactions. Here we used the inherent hydrophobicity and N-glycosylation of membrane proteins to enrich these proteins from the surface and endosome of avian LMH epithelial cells for mass spectrometric analysis. The cycling of many different types of proteins from the cell surface into the endosome and sometimes back to the surface again makes it appropriate to analyze these two membranous cellular components together. Stringent searches of the International Protein Index (IPI) entries for Gallus gallus identified 318 unique integral membrane proteins (IMPs) (201 bearing N-glycosylation sites), 265 unique membrane-associated proteins (MAPs), and an additional group of 784 non-membrane proteins (NMPs) among TX-114 detergent and aqueous phase-enriched proteins. Capture of N-glycosylated tryptic peptides revealed 36 additional glycoproteins most of which were CD antigens, receptors, and molecules for cell adhesion and immune response. IMPs and MAPs present at the surface and within the endosome included proteins involved in transport (255), metabolism (285), communication (108), adhesion (47), and immune responses (42). Among these were 355 putative uncharacterized and hypothetical IMPs, MAPs, and NMPs for which highly similar annotated sequences were found in standard protein–protein BLAST searches.