title: High-throughput identification of IMCD proteins using LC-MS/MS.
abstract: 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. Vasopressin analog (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 one-dimensional 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 gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, 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.
authors.affiliation: Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.forename: Jared
authors.initials: J
authors.lastname: Bieniek
authors.forename: Dmitry
authors.initials: D
authors.lastname: Tchapyjnikov
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.forename: Wells W
authors.initials: WW
authors.lastname: Wu
authors.forename: Rong-Fong
authors.initials: RF
authors.lastname: Shen
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
mesh_terms: D000818:Animals; D050034:Antidiuretic Agents / Q000494:pharmacology; D051399:Aquaporin 2 / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid; D030562:Databases, Protein; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000032:analysis / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D007679:Kidney Medulla / Q000187:drug effects / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics; D050765:Qa-SNARE Proteins / Q000032:analysis / Q000378:metabolism; D051381:Rats; D011910:Rats, Brattleboro; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
chemical_list: D050034:Antidiuretic Agents; C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D053503:Epithelial Sodium Channels; D011506:Proteins; D050765:Qa-SNARE Proteins; D003894:Deamino Arginine Vasopressin