In:
Nephron Physiology, S. Karger AG, Vol. 121, No. 3-4 ( 2013-2-14), p. p9-p15
Abstract:
〈 b 〉 〈 i 〉 Background/Aims: 〈 /i 〉 〈 /b 〉 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. 〈 b 〉 〈 i 〉 Methods: 〈 /i 〉 〈 /b 〉 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). 〈 b 〉 〈 i 〉 Results: 〈 /i 〉 〈 /b 〉 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. 〈 b 〉 〈 i 〉 Conclusion: 〈 /i 〉 〈 /b 〉 We find the method suitable and useful for studying hormonal effects on protein abundance in distal tubular segments.
Type of Medium:
Online Resource
ISSN:
1660-2137
Language:
English
Publisher:
S. Karger AG
Publication Date:
2013
detail.hit.zdb_id:
2098340-2
