Core B: Mass spectrometry, proteomics, metabolomics and lipidomics

项目负责人/主要研究者（最后一名、第一名、中间名）：Kwiatkowski，大卫，J.等人，核心B; Asara 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 （QExactive）质谱和混合三重四极杆（QTRAP）质谱。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， 翻译修饰（PTM）位点作图，如磷酸化、泛素化、乙酰化等，以及 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 除了来自小鼠和人的体内组织来源之外，还将从细胞系、异种移植物进行研究。 肿瘤的我们已经开发了代谢组学分析的专业知识，服务将包括极性代谢物 使用具有极性切换的选择反应监测（SRM）进行分析，以靶向300多个分子， 15分钟。我们将使用稳态分析和13 C和15 N分析细胞、肿瘤组织和生物液体 稳定同位素标记的通量实验，以确定哪些代谢途径在含有 TSC 1/2相关通路的缺陷。还将进行非靶向代谢组学分析，以发现 新的代谢靶点。核心B最近开发了一种非靶向脂质组学平台， 使用新型软件进行极性切换的分辨率质谱法，可识别1000多种脂质离子 （磷脂、甘油三酯、游离脂肪酸等）在不到30分钟。使用反相LC-MS/MS。我们将 还为脂质组学实验开发稳定的同位素通量。除了运行项目1-3的示例之外， B还开发了一种利用单个肿瘤、细胞或体液制备的系列组学技术 用于进行三种不同的组学（整体磷酸化蛋白质组学/蛋白质组学、代谢组学和 脂质组学）。我们还将继续制定经济学战略， 将模型物种（果蝇）与癌细胞和肿瘤组织重叠，以揭示保守的生物学相互作用 TSC 1/2和相关通路中的潜在生物标志物靶点。

Program Director/Principal Investigator (Last, First, Middle): Kwiatkowski, David, J. et al., Core B; Asara 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 (QExactive) mass spectrometry and hybrid triple quadrupole (QTRAP) mass spectrometry. For proteomics, microcapillary tandem mass spectrometry (LC-MS/MS) services will include protein complex identification, 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 mice and human tumors. 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 will also be performed to discover novel metabolic targets. Core B has recently developed a non-targeted lipidomics platform based on high resolution mass spectrometry with polarity switching with novel software to identify more than 1000 lipid ions (phospholipids, triglycerides, free fatty acids, etc.) in less than 30 min. using reversed-phase LC-MS/MS. We will also develop stable isotope flux for lipidomics experiments. In addition to running samples for Projects 1-3, Core B has also developed a serial-omics technology that utilizes the preparation of a single tumor, 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.