BACKGROUND/AIMS: 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. CONCLUSION: We find the method suitable and useful for studying hormonal effects on protein abundance in distal tubular segments.
BACKGROUND/AIMS: 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. CONCLUSION: We find the method suitable and useful for studying hormonal effects on protein abundance in distal tubular segments.
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