The Mass Spectrometry and Proteomics Core will provide protein identification, phosphorylation sitemapping, and develop strategies for relative quantification of proteins/phosphopeptides in order to aid in theunderstanding of the Tuberous sclerosis pathway and pathogenesis, LKB1/AMPK signaling and its role inPeutz-Jeghers syndrome, and a dissection of Tsc1/Tsc2/Tor/S6K signaling. Liquid chromatography coupledto tandem mass spectrometry (LC/MS/MS) will be used to identify proteins and phosphorylation sites as wellas provide quantitative information for proteins and phosphorylation sites. Peptides from tryptic digestionswill be analyzed by microcapillary reversed-phase LC/MS/MS using a LTQ linear ion trap mass spectrometerand a QSTAR Pulsar quadrupole-TOF mass spectrometer. Proteins and their phosphorylation sites will beidentified after gel separation by interrogating non-redundant protein databases with peptide tandem massspectra. Relative quantitation of proteins will be performed by isotopically labeling the proteins from differentcell states with light and heavy amino acids, respectively, using stable isotope labeling of amino acids in cellculture (SILAC) using the QSTAR mass spectrometer or labeling post cell growth at the protein and/orpeptide level using global internal standard technology (GIST) primary amine stable isotope labelingreagents. Ratios of light to heavy peptide pairs will be determined using both MSQuant and in-housedeveloped software. Bioinformatics will be used for data management and data mining for biologicalsignificance.
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