title: Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions.
abstract: The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out "deep" proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate "virtual Western blots" for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa.
authors: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and.
authors: Chin-Rang
authors: CR
authors: Yang
authors: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and Inter-Department Program of Biomedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok, Thailand.
authors: Pumipat
authors: P
authors: Tongyoo
authors: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and.
authors: Milad
authors: M
authors: Emamian
authors: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and.
authors: Pablo C
authors: PC
authors: Sandoval
authors: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and.
authors: Viswanathan
authors: V
authors: Raghuram
authors: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and knepperm@nhlbi.nih.gov.
authors: Mark A
authors: MA
authors: Knepper
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002460:Cell Line / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells; D007685:Kidney Tubules, Collecting; D051379:Mice; D008970:Molecular Weight; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014667:Vasopressins / Q000378:metabolism
chemical_list: D014667:Vasopressins
keywords: aquaporin-2; differential centrifugation; liquid chromatography-tandem mass spectrometry; mass spectrometry; mpkCCD