Quantitative large‐scale phosphotyrosine proteomics of vasopressin‐sensitive inner medullary collecting duct cells using IVICAT N‐terminal labeling and LC‐MS/MS

Abstract

Large-scale identification of phosphotyrosyl (pTyr)-proteins by LC-MS methods remains challenging. We previously developed a novel isotopic labeling method, called IVICAT (In Vacuo Isotope-Coded Alkylation Technique) that improves the detection sensitivity of pTyr-containing peptides in positive ion ESI-MS by trimethylating the N-terminus of every peptide, thereby creating a fixed positive charge. Here we use pTyr immunoprecipitation, IVICAT labeling, IMAC, and LC-MS/MS to profile the pTyr proteome of inner medullary collecting duct (IMCD) cells isolated from rat kidneys. These experiments identified 114 pTyr sites, 103 of which have not been previously reported. Using H3- or D3-methylating reagents in IVICAT, we quantified pTyr changes in response to the V2 receptor selective analog dDAVP in IMCD cell suspensions. Use of our in house software (QUIL) for construction of pseudochromatograms from time-dependent LC-MS data resulted in the discovery of several sites modified by vasopressin including phospholipase C gamma-1 (PLCγ1) at Y771 (increase of approximately two-fold confirmed by immunoblotting). Differential centrifugation showed that dDAVP displaces PLCγ1 from membrane to cytosol, consistent with a net inhibitory action. The data indicate that vasopressin signaling includes changes in tyrosine phosphorylation that have potential roles in regulation of aquaporins or urea transporters.