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1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institutes, National Institutes of Health, Bethesda, MD, USA
2 Proteomics Core Facility, National Heart, Lung, and Blood Institutes, National Institutes of Health, Bethesda, MD, USA
* To whom correspondence should be addressed. E-mail: knepperm{at}nhlbi.nih.gov.
The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell, and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database", containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. dDAVP or vehicle was infused subcutaneously in Brattleboro rats for 3 days and IMCDs were isolated for proteomic analysis. dDAVP and control samples were
labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by 1-D SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification based on semiquantitative immunoblotting of 16 proteins for which antibodies were available showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and 
-ENaC, five of the immunoblotted proteins were substantially altered in abundance in response to
dDAVP, viz. syntaxin-7, Rap1, GAPDH, HSP70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.
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