LC‐MS/MS‐based large‐scale profiling of protein half lives in renal collecting duct cells reveals that vasopressin increases half‐life of aquaporin‐2 protein

Abstract

Long-term exposure of collecting duct cells to vasopressin increases the abundance of aquaporin-2 (AQP2) as well as that of other proteins. To discover mechanisms involved, we carried out large-scale measurements of protein half-lives (t1/2) in cultured renal mpkCCD(clone 11) cells at steady state using metabolic labeling with stable isotopes (SILAC) coupled with protein mass spectrometry (LC-MS/MS). A mass-balance model assuming first-order degradation kinetics, allowed calculation of t1/2 for over 3000 proteins. The shortest half lives were found for proteins associated with Gene Ontology terms “cell junction” and “endosome”. Half lives for over 1000 proteins were quantified in each of 3 pairs of experiments (n=3) (± dDAVP, 1 nM). 108 were found to change significantly in response to vasopressin. Among the proteins with regulated t1/2 was AQP2, which underwent a significant increase from a mean of 10.2 to 14.1 hours (P<0.005, paired t-test), explaining part of the observed increase in AQP2 abundance. AQP2 is found in two bands (at 29 kDa and 37 kDa) in immunoblots. Using t1/2 data from SDS-PAGE-separated proteins, we found that AQP2 in the upper band has a significantly lower t1/2 than does AQP2 in the lower band (12.4 hr vs 15.1 hr, P<0.02), presumably due to the post-translational modifications that account for the higher molecular weight.