Core B: Mass Spectrometry, proteomics, metabolmics and lipidomics

点击翻译按钮获取中文摘要

Core B: Project Summary/Abstract The mass spectrometry core has expertise in proteomics/phosphoproteomics, metabolomics and lipidomics resources to enable the three major P01 projects achieve success in uncovering the molecular mechanisms of Hamartoma syndromes and related cancers in the TSC1-TSC2 pathways for new drug targets and novel therapies using tandem mass spectrometry (LC-MS/MS). The core utilizes both high resolution hybrid Orbitrap (Exploris 480, QExactive HF) mass spectrometry and hybrid triple quadrupole (QTRAP 6500/5500) mass spectrometry. For proteomics, microcapillary tandem mass spectrometry (LC-MS/MS) services will include protein complex identification, global post-translational modification (PTM) site mapping such as phosphorylation, ubiquitination, acetylation, etc. and the relative and absolute quantification of peptides/proteins using both stable isotope labeling (SILAC and TMT) and label-free quantification [spectral counting, total ion current (TIC), multiple reaction monitoring (MRM)]. These studies will be performed from cell lines, xenografts in addition to in vivo tissue sources from mouse/human tumors and drosophila models. We have developed expertise in metabolomics profiling and services will include polar metabolite profiling using selected reaction monitoring (SRM) with polarity switching to target more than 300 molecules in 15 min. We will profile cells, tumor tissues and biological fluids using both steady-state profiling and 13C and 15N stable isotope labeled flux experiments to determine which metabolic pathways are altered in cells harboring defects in the TSC1/2 related pathways. Non-targeted metabolomic profiling by HR-LC-MS/MS will also be performed to discover novel metabolic targets. Core B will use non-targeted lipidomics based on high resolution mass spectrometry with polarity switching with novel software to identify more than 1500 lipid ions (phospholipids, triglycerides, free fatty acids, etc.) in less than 30 min. using reversed-phase LC-MS/MS. We will also use recently developed stable 13C/15N/18O isotope flux for lipidomics. In addition to running samples for Projects 1-3, Core B has developed a serial-omics technology that utilizes the preparation of a single tisue, cell or bodily fluid sample for performing three different –omics (global phosphoproteomics/proteomics, metabolomics and lipidomics) via partitioning liquid-liquid extraction layers. We will also continue to develop -omics strategies to overlap model species (drosophila) to cancer cells and tumor tissue to uncover conserved biological interactions for potential biomarker targets in TSC1/2 and related pathways.