Core B: Mass Spectrometry, proteomics, metabolmics and lipidomics

核心B：项目总结/摘要 质谱核心在蛋白质组学/磷酸化蛋白质组学、代谢组学和脂质组学方面具有专长 资源，使三个主要的P01项目取得成功，揭示分子机制， 错构瘤综合征和相关癌症的新药物靶点和新的TSC 1-TSC 2途径 使用串联质谱法（LC-MS/MS）对治疗进行评价。核心利用高分辨率混合轨道阱和 （Exploris 480，QExactive HF）质谱和混合三重四极杆（QTRAP 6500/5500）质谱 光谱法对于蛋白质组学，微毛细管串联质谱（LC-MS/MS）服务将包括 蛋白质复合物鉴定、全局翻译后修饰（PTM）位点作图， 磷酸化、泛素化、乙酰化等以及肽/蛋白质的相对和绝对定量 使用稳定同位素标记（SILAC和TMT）和无标记定量[光谱计数，总离子 电流（TIC），多反应监测（MRM）]。这些研究将从细胞系、异种移植物、 除了来自小鼠/人肿瘤和果蝇模型的体内组织来源之外。我们已经开发 代谢组学分析和服务方面的专业知识将包括使用选定反应的极性代谢物分析 我们将在15分钟内通过极性切换监测（SRM）靶向300多个分子。 使用稳态分布和13 C和15 N稳定同位素标记通量的组织和生物流体 实验以确定在TSC 1/2相关缺陷的细胞中哪些代谢途径被改变， 途径。还将通过HR-LC-MS/MS进行非靶向代谢组学分析，以发现新的 代谢目标。核心B将使用基于高分辨率质谱的非靶向脂质组学， 极性切换与新的软件，以确定超过1500个脂质离子（磷脂，甘油三酯，游离脂肪 酸等）使用反相LC-MS/MS。我们还将使用最近开发的稳定的 用于脂质组学的13 C/15 N/18 O同位素通量。除了运行项目1-3的示例外，核心B还开发了 一种系列组学技术，其利用单个组织、细胞或体液样品的制备来进行 三个不同的组学（整体磷酸化蛋白质组学/蛋白质组学、代谢组学和脂质组学） 液-液萃取层。我们还将继续开发组学策略，以重叠模式物种 （果蝇）到癌细胞和肿瘤组织，以揭示潜在生物标志物的保守生物相互作用 TSC 1/2和相关通路的靶点。

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.