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