This database provides phosphopeptide ratios of desmopressin (dDAVP)-treated and untreated PKA-null mpkCCD collecting duct cells, determined using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and an Orbitrap Fusion Lumos Tribrid mass spectrometer. PKA-null cells were developed from mpkCCD cells by deleting both PKA catalytic subunit genes via CRISPR-Cas9 (PMID: 28973931). 

Brief Method: For short-term dDAVP stimulation, three independent clones of PKA-null 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. Phosphosite was designated with * (site assignment probability , >80%) or with ? (<80%). P_fp, probability of false positive ratio difference.

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Reference: Datta A, Yang CR, Limbutara K, Chou CL, Rinschen MM, Raghuram V, Knepper MA. PKA-independent vasopressin signaling in renal collecting duct. FASEB J. 2020;34:6129-6146. (PMID: 32219907).

The website was created by Arnab Datta, Anika Kao, Chin-Rang Yang and Mark A. Knepper. Please contact Mark A. Knepper for comments or questions: knep@helix.nih.gov.

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