title: Large-scale phosphoproteomic analysis of membrane proteins in renal proximal and distal tubule.
abstract: Recent advances in mass spectrometry (MS) have provided means for large-scale phosphoproteomic profiling of specific tissues. Here, we report results from large-scale tandem MS [liquid chromatography (LC)-MS/MS]-based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, with focus on transporters and regulatory proteins. Data sets were filtered (by target-decoy analysis) to limit false-positive identifications to <2%. A total of 7,125 unique nonphosphorylated and 743 unique phosphorylated peptides were identified. Among the phosphopeptides identified were sites on transporter proteins, i.e., solute carrier (Slc, n = 63), ATP-binding cassette (Abc, n = 4), and aquaporin (Aqp, n = 3) family proteins. Database searches reveal that a majority of the phosphorylation sites identified in transporter proteins were previously unreported. Most of the Slc family proteins are apical or basolateral transporters expressed in proximal tubule cells, including proteins known to mediate transport of glucose, amino acids, organic ions, and inorganic ions. In addition, we identified potentially important phosphorylation sites for transport proteins from distal nephron segments, including the bumetanide-sensitive Na-K-2Cl cotransporter (Slc12a1 or NKCC2) at Ser(87), Thr(101), and Ser(126) and the thiazide-sensitive Na-Cl cotransporter (Slc12a3 or NCC) at Ser(71) and Ser(124). A subset of phosphorylation sites in regulatory proteins coincided with known functional motifs, suggesting specific regulatory roles. An online database from this study (http://dir.nhlbi.nih.gov/papers/lkem/rcmpd/) provides a resource for future studies of transporter regulation.
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Marina
authors.initials: M
authors.lastname: Feric
authors.forename: Boyang
authors.initials: B
authors.lastname: Zhao
authors.forename: Jason D
authors.initials: JD
authors.lastname: Hoffert
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D006801:Humans; D007686:Kidney Tubules, Distal / Q000737:chemistry* / Q000166:cytology; D007687:Kidney Tubules, Proximal / Q000737:chemistry* / Q000166:cytology; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000235:genetics; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry
chemical_list: D008565:Membrane Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome