Nuclear Proteomics of the Inner Medullary Collecting Duct (IMCD)

Abstract

Mechanisms of vasopressin‐induced transcriptional regulation in the IMCD are poorly understood. The low abundance of many transcriptional regulators has been a limiting factor in their identification in proteomic studies. To address this, we developed an approach for the purification and enrichment of IMCD nuclear proteins based on differential centrifugation and detergent extraction. Immunoblotting demonstrated a forty‐fold enrichment of the nuclear marker BRG1, while the cytosolic marker aldose reductase was de‐enriched fifty‐fold and the membrane marker AQP2 was de‐enriched ten‐fold. We used 1‐D SDS‐PAGE LC‐MS/MS to identify IMCD nuclear proteins. Two types of protein identification algorithms were employed (SEQUEST and InsPecT), both coupled to a target‐decoy methodology to limit false‐positive identifications to less than 2%. We also designed a suite of JAVA‐based bioinformatics software, called APIEX, for the filtering, analysis, and annotation of these proteomic data sets. With this approach, we identified over 2300 proteins, including many transcription factors with potential relevance to vasopressin signaling (including CREB1, GATA3, STAT1, PAX8, NF‐κB, YY1, KRL7a, Smad 1, and Smad 4). The identified proteins add to the known IMCD proteome (http://dir.nhlbi.nih.gov/papers/lkem/imcd/) and lay the groundwork for a more comprehensive systems biology study of the collecting duct.