title: Assessment of the effect of 24-hour aldosterone administration on protein abundance in fluorescence-sorted mouse distal renal tubules by mass spectrometry.
abstract: OBJECTIVE
  Aldosterone exerts multiple long-term effects on the distal renal tubules. The aim of this study was to establish a method for identifying proteins in these tubules that change in abundance by only 24-hour aldosterone administration.
  
  
  METHODS
  Mice endogenously expressing green fluorescent protein (eGFP) in the connecting tubule and cortical collecting ducts were treated with a subcutaneous injection of 2.0 mg/kg aldosterone or vehicle (n = 5), and sacrificed 24 h later. Suspensions of single cells were obtained enzymatically, and eGFP-positive cells were isolated by fluorescence-activated cell sorting (FACS). Samples of 100 µg of proteins were digested with trypsin and labeled with 8-plex isobaric tags for relative and absolute quantitation reagents and processed for liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  
  
  RESULTS
  FACS yielded 1.4 million cells per mouse. The LC-MS/MS spectra were matched to peptides by the SEQUEST search algorithm, which identified 3,002 peptides corresponding to 506 unique proteins, of which 20 significantly changed abundance 24 h after aldosterone injection.
  
  
  CONCLUSIONS
  We find the method suitable and useful for studying hormonal effects on protein abundance in distal tubular segments.
authors: Department of Biomedicine and the Water and Salt Research Center, Aarhus University - Health, Aarhus, Denmark.
authors: Thomas B
authors: TB
authors: Jensen
authors: Trairak
authors: T
authors: Pisitkun
authors: Jason D
authors: JD
authors: Hoffert
authors: Uffe B
authors: UB
authors: Jensen
authors: Robert A
authors: RA
authors: Fenton
authors: Helle A
authors: HA
authors: Praetorius
authors: Mark A
authors: MA
authors: Knepper
authors: Jeppe
authors: J
authors: Praetorius
mesh_terms: D000450:Aldosterone / Q000008:administration & dosage* / Q000097:blood; D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002853:Chromatography, Liquid; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007279:Injections, Subcutaneous; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010449:Peptide Mapping; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D011506:Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000450:Aldosterone