This database provides phosphopeptide ratios of desmopressin (dDAVP)-treated and untreated mpkCCD collecting duct cells, determined using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and an Orbitrap Fusion Lumos Tribrid mass spectrometer.

Brief Method: For short-term dDAVP stimulation, three independent clones of mpkCCD cells were maintained in the absence of dDAVP for 2 h (wash-out phase) on the day of experiment, and were then treated with either 0.1 nM dDAVP or vehicle for 30 min on the basolateral side only. The proteins were extracted, mixed (total = 2mg), reduced, alkylated and digested with Trypsin/LysC. Both Fe-NTA and TiO₂ columns (Thermo Fisher) were used for parallel enrichment of phosphopeptides. Protein mass spectrometry of each sample was performed using both HCD and EThcD fragmentation.

Data Analysis: Proteome Discoverer 1.4 (version 1.4.0.288) using two search engines: Mascot and SequestHT [mouse Swiss-Prot (April 24, 2017)]. Data were filtered to eliminate low abundance phosphopeptides based on MS1 AUC. Phosphosites were designated with " * ". CV, Co-efficient of Variation.

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Reference: Datta A, Yang CR, Salhadar K, Park E, Chou CL, Raghuram V, Knepper MA. Phosphoproteomic identification of vasopressin-regulated protein kinases in collecting duct cells. Br J Pharmacol. 2021 Mar;178(6):1426-1444.

The website was created by Arnab Datta and Mark A. Knepper. Please contact Mark A. Knepper for comments or questions: knep@helix.nih.gov.

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