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Originally published In Press as doi:10.1074/mcp.M600177-MCP200 on August 9, 2006.
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Molecular & Cellular Proteomics 5:2131-2145, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

LC-MS/MS Analysis of Apical and Basolateral Plasma Membranes of Rat Renal Collecting Duct Cells*,S

Ming-Jiun Yu{ddagger}, Trairak Pisitkun{ddagger}, Guanghui Wang§, Rong-Fong Shen§ and Mark A. Knepper{ddagger},¶

From the {ddagger} Laboratory of Kidney and Electrolyte Metabolism and § Proteomics Core Facility, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

We used biotinylation and streptavidin affinity chromatography to label and enrich proteins from apical and basolateral membranes of rat kidney inner medullary collecting ducts (IMCDs) prior to LC-MS/MS protein identification. To enrich apical membrane proteins and bound peripheral membrane proteins, IMCDs were perfusion-labeled with primary amine-reactive biotinylation reagents at 2 °C using a double barreled pipette. The perfusion-biotinylated proteins and proteins bound to them were isolated with CaptAvidin-agarose beads, separated with SDS-PAGE, and sliced into continuous gel pieces for LC-MS/MS protein identification (LTQ, Thermo Electron Corp.). 17 integral and glycosylphosphatidylinositol (GPI)-linked membrane proteins and 44 non-integral membrane proteins were identified. Immunofluorescence confocal microscopy confirmed ACVRL1, H+/K+-ATPase {alpha}1, NHE2, and TauT expression in the IMCDs. Basement membrane and basolateral membrane proteins were biotinylated via incubation of IMCD suspensions with biotinylation reagents on ice. 23 integral and GPI-linked membrane proteins and 134 non-integral membrane proteins were identified. Analyses of non-integral membrane proteins preferentially identified in the perfusion-biotinylated and not in the incubation-biotinylated IMCDs revealed protein kinases, scaffold proteins, SNARE proteins, motor proteins, small GTP-binding proteins, and related proteins that may be involved in vasopressin-stimulated AQP2, UT-A1, and ENaC regulation. A World Wide Web-accessible database was constructed of 222 membrane proteins (integral and GPI-linked) from this study and prior studies.

To whom correspondence should be addressed: National Institutes of Health, Bldg. 10, Rm. 6N260, 10 Center Dr., MSC-1603, Bethesda, MD 20892-1603. Tel.: 301-496-3064; Fax: 301-402-1443; E-mail: knep{at}helix.nih.gov


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