The mTAL Phosphoproteome Database
contains phosphoprotein mass
spectrometry data from native renal medullary thick ascending limb (mTAL)
-enriched tubule suspensions. The database was created by Ruwan Gunaratne, Trairak
Pisitkun, and Mark Knepper of the Epithelial Systems Biology Laboratory, Division
of Intramural Research, NHLBI, NIH. Bethesda.Please cite:Gunaratne R,
Braucht DW, Rinschen MM, Chou CL, Hoffert JD, Pisitkun T, Knepper MA.
Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent
signaling pathways in native renal thick ascending limb cells. Proc Natl Acad Sci U S A.
2010;107:15653-8.PMID: 20713729. Contact us for questions or comments.
Large-scale quantitative phosphoproteomic profiling was performed on paired
vehicle- and hormone-treated mTAL-enriched suspensions. Hormone treatment utilized a mixture of hormones that increase cAMP levels (vasopressin, glucagon, parathyroid hormone, and calcitonin) through GPCRs. The samples were
analyzed on an LTQ-Orbitrap mass spectrometer and 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. Quantification of
phosphopeptide abundance (area under the curve of the reconstructed ion
chromatogram elution profile) was achieved using QUOIL, an in-house software
program designed for quantification of label-free peptides by LC-MS (NHLBI
Proteomics Core Facility). As a result, 654 unique phosphopeptides
corresponding to 374 unique phosphoproteins were identified.