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This Renal Cortical Membrane Phosphoproteome Database is based on protein mass spectrometry data from the NHLBI Epithelial Systems Biology Laboratory. All data are from membrane fractions enriched from native rat cortex by the method of Biber et al. with modifications. These samples are predominated by apical and basolateral plasma membrane proteins from proximal tubule as well as other nephron segments including thick ascending limb and distal convoluted tubule.

Experimental description:

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.

The Renal Cortical Membrane Phosphoproteome Database was created by Marina Feric, Boyang Zhao, Jason Hoffert, Trairak Pisitkun, and Mark Knepper.

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Phosphopeptide Sequence Site Assignment: * = specific phosphosite assigned, ? = site assignment ambiguous, @ = oxidation

1 Frequency: The total amount of scans that produce the same phosphopeptide sequence

2 Specific Site Assignment: The certainty of the phosphorylation site is "yes" if the phosphopeptide passed: 1) the Ascore and/or the PhosphoScore for the SEQUEST search algorithm; and/or 2) the Phosphate Localization score (PLS) for the InsPecT search algorithm. The certainty of the phosphorylation is "no" if: 1) there was a discrepancy among the various search algorithms; 2) the peptide was not tested for the Ascore, PhosphoScore, or PLS (peptide identified solely by OMSSA search algorithm); or 3) the peptide failed to pass the Ascore, PhosphoScore, or PLS. Thus, the phosphoylation site is ambiguous when the certainty is "no".