Deep Proteomic Quantification of Inner Medullary Collecting Duct Protein Phosphorylation: Response to Vasopressin

Abstract

Progress in protein mass spectrometry instrumentation and labeling methods have led to dramatic increases in the depth of phospho‐peptide identification and quantification. Here we used tandem mass tag (TMT) labeling and LC‐MS/MS for deep identification of phospho‐proteomic responses to the vasopressin analog dDAVP (1 nM for 30 minutes) in inner medullary collecting duct (IMCD) suspensions from rats. We analyzed three pairs of samples (dDAVP vs. vehicle) on different days. The analysis yielded 16 GB of spectral data mapping to 11,658 phospho‐peptides in 1758 distinct proteins. Vehicle vs. vehicle comparisons allowed assessment of the variability of the method and permitted dual criteria to be applied for stringent identification of abundance changes (based on t‐statistic and z‐score). Among all phospho‐peptides quantified, only 93 were increased in abundance, and only 49 were decreased, indicating very selective regulation within the broad range of phospho‐peptides quantified. A substantial number of the increases were in aquaporin‐2 and the UT‐A urea channel, including a previously unidentified phosphorylation site at Ser918 of UT‐A1 (sequence RAS*MITK) that was more than doubled in abundance. Abundance changes in aquaporin‐2 peptides confirmed prior findings. Of interest, phosphorylation changes in Raf1 (increased phosphorylation at Ser259, a kinase inhibitory site) and A‐Raf (decreased phosphorylation of Ser255, a kinase activating site) provide an explanation for findings that vasopressin decreases ERK activation and AQP2 phosphorylation at Ser261 in the collecting duct. Finally, vasopressin significantly increased phosphorylation of protein kinase D1 (Prkd1) at Ser255 and of phosphatidylinositol 4‐kinase beta at Ser551. Both proteins play central roles in regulated exocytosis of ENaC, and we speculate that they could play similar roles in vasopressin‐stimulated exocytotic apical insertion of aquaporin‐2 and UT‐A1.Support or Funding InformationV.D. was a member of the 2017 Biomedical Engineering Student Internship Program at the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.