Brush border membrane-enriched samples from renal cortices of male Sprague-Dawley rats (6-8 weeks old) were obtained via MgCl₂ precipitation and differential centrifugation according to established protocols (Biber et al.). Protein samples were subjected to in-solution trypsin digestion followed by SCX and IMAC for phosphoproteomic profiling using a Thermo LTQ-FT mass spectrometer. The resulting MS spectra were searched using three search algorithms: SEQUEST, InsPecT, and OMSSA. In all cases, a target-decoy analysis approach was undertaken to limit the false discovery rate to < 2%. Phosphorylation site assignment was carried out using the following scoring algorithms: Ascore and PhosphoScore for SEQUEST data, and Phosphate Localization Score for InsPecT data. In order to assign a phosphorylation site correctly, at least one score had to pass the given threshold (Ascore = 19, PhosphoScore was "Passed" or "OneChoice", PLS score = 8). Final assignment of phosphorylation sites was performed using the in-house programs, NHLBI Promatch and Phosphosite.