Proteomic profiling of nuclei from native renal inner medullary collecting duct cells using LC-MS/MS

Abstract

Vasopressin is a peptide hormone that regulates renal water excretion in part through its actions on the collecting duct. The regulation occurs in part via control of transcription of genes coding for the water channels aquaporin-2 ( Aqp2) and aquaporin-3 ( Aqp3). To identify transcription factors expressed in collecting duct cells, we have carried out LC-MS/MS-based proteomic profiling of nuclei isolated from native rat inner medullary collecting ducts (IMCDs). To maximize the number of proteins identified, we matched spectra to rat amino acid sequences using three different search algorithms (SEQUEST, InsPecT, and OMSSA). All searches were coupled to target-decoy methodology to limit false-discovery identifications to 2% of the total for single-peptide identifications. In addition, we developed a computational tool ( ProMatch) to identify and eliminate ambiguous identifications. With this approach, we identified >3,500 proteins, including 154 proteins classified as “transcription factor” proteins (Panther Classification System). Among these, are members of CREB, ETS, RXR, NFAT, HOX, GATA, EBOX, EGR, MYT1, KLF, and CP2 families, which were found to have evolutionarily conserved putative binding sites in the 5′-flanking region or first intron of the Aqp2 gene, as well as members of EBOX, NR2, GRE, MAZ, KLF, and SP1 families corresponding to conserved sites in the 5′-flanking region of the Aqp3 gene. In addition, several novel phosphorylation sites in nuclear proteins were identified using the neutral loss-scanning LC-MS3technique. The newly identified proteins have been incorporated into the IMCD Proteome Database ( http://dir.nhlbi.nih.gov/papers/lkem/imcd/ ).