Large‐scale phosphoproteomic profiling of native rat renal collecting duct epithelium: Tyrosine phosphorylation

Abstract

To systematically identify the phosphotyrosine (pY) sites in the renal collecting duct epithelium, we used LC‐MS/MS (LTQ‐Orbitrap) after pY peptide enrichment. Suspensions of renal inner medullary collecting ducts (IMCDs) from rat kidneys were pre‐incubated for 10 min with a physiological saline solution to which was added 0.1 mM sodium pervanadate to maximize tyrosine phosphorylation. Isolated proteins were subjected to pY immunoprecipitation followed by Ga3+‐immobilized metal affinity chromatography. Using target‐decoy analysis to limit false positives to <1%, we identified 503 unique pY peptides, corresponding to 273 phosphoproteins, which have been added to the online IMCD Phosphoproteome Database (http://goo.gl/oYKu). Site/sequence conservation analysis using custom software indicated that over 84% of the sites are highly conserved, suggesting functional roles. Analysis of the pY‐containing protein set relative to the whole IMCD transcriptome showed highly significant enrichment of proteins present in the KEGG “Adherens Junction” Pathway and highly significant enrichment of proteins with the following Gene Ontology Biological Process terms: “cell adhesion”, “actin cytoskeletal organization”, and “cell migration”. These associations point to likely roles of tyrosine phosphorylation‐dependent signaling pathways in the renal collecting duct epithelia. (BZ was a 2010 NIBIB‐BESIP summer intern from the U. of Michigan.)