Large-Scale Phosphoproteomic Analysis of Membrane Proteins in Renal Proximal and Distal Tubule

Marina Feric, Boyang Zhao, Jason D. Hoffert, Trairak Pisitkun, Mark A. Knepper


Recent advances in mass spectrometry have provided means for large-scale phosphoproteomic profiling of specific tissues. Here, we report results from large-scale LC-MS/MS-based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, focusing on transporters and regulatory proteins. Data sets were filtered (target-decoy) to limit false-positive identifications to <2%. A total of 7,125 unique non-phosphorylated and 743 unique phosphorylated peptides were identified. The phosphopeptides that were found included sites on transporter proteins, i.e. in Slc (n=63), Abc (n=4), and Aqp (n=3) family proteins. A majority of the phosphorylation sites identified in transporter proteins were previously unreported, based on database searches. 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 Ser87, Thr101 and Ser126 and the thiazide-sensitive Na:Cl cotransporter (Slc12a3 or NCC) at Ser71 and Ser124. A subset of phosphorylation sites in regulatory proteins coincided with known functional motifs suggesting specific regulatory roles. An online database from this study (URL: provides a resource for future studies of transporter regulation.

  • mass spectrometry
  • kidney
  • thiazide
  • Slc12 Protein Family
  • kinase