title: Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex.
abstract: Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.
authors.affiliation: Ludwig Institute for Cancer Research, University College London Branch, London W1W 7BS, UK. pedro@ludwig.ucl.ac.uk
authors.forename: Pedro R
authors.initials: PR
authors.lastname: Cutillas
authors.forename: Juerg
authors.initials: J
authors.lastname: Biber
authors.forename: Joanne
authors.initials: J
authors.lastname: Marks
authors.forename: Richard
authors.initials: R
authors.lastname: Jacob
authors.forename: Bruno
authors.initials: B
authors.lastname: Stieger
authors.forename: Rainer
authors.initials: R
authors.lastname: Cramer
authors.forename: Michael
authors.initials: M
authors.lastname: Waterfield
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Unwin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D016764:Cell Polarity; D002498:Centrifugation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007672:Kidney Cortex / Q000737:chemistry*; D008871:Microvilli / Q000737:chemistry; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D020543:Proteome