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.
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.
Conclusion
We find the method suitable and useful for studying hormonal effects on protein abundance in distal tubular segments.
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.