Core B: Mass Spectrometry, proteomics, metabolmics and lipidomics

核心B：项目摘要/摘要 质谱学核心在蛋白质组学/磷蛋白质组学、代谢组学和脂类组学方面拥有专业知识。 资源使三个重大P01项目成功地揭示了 错构瘤综合征和TSC1-TSC2通路中的相关癌症寻找新的药物靶点和新药物 使用串联质谱仪(LC-MS/MS)进行治疗。核心利用了两个高分辨率混合轨道捕获 (Exploris 480，QExactive HF)质谱学和混合三重四极杆(QTRAP 6500/5500)质量 光谱分析。在蛋白质组学方面，微毛细管串联质谱仪(LC-MS/MS)服务将包括 蛋白质复合体鉴定、全球翻译后修饰(PTM)位点定位，例如 磷酸化、泛素化、乙酰化等与多肽/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 除了来自鼠/人肿瘤和果蝇模型的体内组织来源。我们已经开发出 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 监测(SRM)与极性切换目标超过300个分子在15分钟内。我们将分析细胞、肿瘤 使用稳态分析和13C和15N稳定同位素标记通量的组织和生物液 确定TSC1/2相关缺陷细胞中哪些代谢途径发生改变的实验 小路。还将通过HR-LC-MS/MS进行非靶向代谢组谱分析，以发现新的 代谢目标。核心B将使用基于高分辨率质谱学的非靶向脂质组学 使用新型软件进行极性切换，识别1500多种脂类离子(磷脂、甘油三酯、游离脂肪 酸等)在不到30分钟内。使用反相LC-MS/MS我们还将使用最近开发的稳定剂 脂肪组学的13C/15N/18O同位素通量。除了为项目1-3运行样本外，核心B还开发了 一种系列组学技术，它利用单个组织、细胞或体液样本的制备来进行 三种不同的组学(全球磷蛋白质组学/蛋白质组学、代谢组学和脂类组学)通过分配 液-液萃取层。我们还将继续开发组学战略，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记物的保守生物相互作用 TSC1/2及其相关通路中的靶点。

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.