Quantitative proteomic analysis of long‐term vasopressin actions in mpkCCD cells using SILAC and MRM

Abstract

We employed 'stable isotope labeling with amino acids in cell culture' (SILAC) coupled LC‐MS/MS to identify proteins that change in abundance in response to long‐term vasopressin stimulation in mpkCCD cells. Cells were grown in media containing heavy isotope‐substituted or light non‐substituted amino acids, and either vasopressin analog dDAVP (100pM) or vehicle, respectively. After 5 days, heavy‐ and light‐labeled cell lysates were combined for quantitation by LC‐MS/MS. 65 proteins were increased and 56 were decreased in abundance in response to dDAVP. Increases in 5 proteins from the SILAC results were confirmed by immunoblotting: AQP2, ADD1 (adducin‐1), MAL2, AKAP12, and SLC9A3R1 (NHERF1). For antibody‐independent validation, proteins were selected for targeted quantitative proteomics using multiple‐reaction monitoring (MRM) on a triple quadrupole mass spectrometer. Increases in 4 proteins (AQP2, ASAP2 [ArfGAP with SH3 domain ankyrin repeat and PH domain 2], MON2 [yeast homolog involved in Golgi to endosome transport] and CLMN [calmin]) and a decrease in one protein (NALCN [sodium leak channel non‐selective]) were statistically validated. Overall 1,629 proteins were identified and deposited in a new online database (mpkCCD Proteome Database). These methodologies permit large‐scale targeted quantification, providing an antibody‐independent alternative to multiplexed immunoblotting.