Core B: Mass spectrometry, proteomics, metabolomics and lipidomics

核心 B：质谱、蛋白质组学、代谢组学和脂质组学

• 批准号: 10221617
• 负责人: John M Asara
• 金额: $ 17.41万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221617
• 关键词: Acetylation; Biological; Biological Models; Biological Process; Biopsy; Cell Line; Cells; Computer software; Data; Defect; Development; Drosophila genus; Drug Targeting; Elements; Energy-Generating Resources; Hamartoma; Human; Hybrids; Ions; Label; Libraries; Lipids; Liquid substance; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Metabolic Pathway; Methods; Modeling; Molecular; Monitor; Multiple Myeloma; Mus; Nonesterified Fatty Acids; Pathogenesis; Pathway interactions; Peptides; Phase; Phospholipids; Phosphorylation; Post-Translational Modification Site; Preparation; Principal Investigator; Proteins; Proteome; Proteomics; Reaction; Resolution; Resources; Running; Sampling; Serum; Services; Source; Stable Isotope Labeling; Syndrome; System; TERT gene; TSC1 gene; TSC1/2 gene; TSC2 gene; Technology; Testing; Tissues; Triglycerides; Tuberous sclerosis protein complex; Tumor Tissue; Ubiquitination; Vendor; Washington; Xenograft procedure; base; cancer cell; experimental study; in vivo; instrumentation; lipidomics; mass spectrometer; metabolomics; multiple reaction monitoring; neoplastic cell; new technology; new therapeutic target; novel; novel therapeutics; phosphoproteomics; potential biomarker; programs; protein complex; scaffold; small molecule; stable isotope; stem; success; tandem mass spectrometry; targeted biomarker; targeted treatment; tumor

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.

项目主任/首席调查员(最后、第一、中间)：Kwiatkowski，David，J.等，核心B；Asara 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 (QExactive)质谱仪和混合三重四极杆(QTRAP)质谱仪。对于蛋白质组学来说， 微毛细管串联质谱(LC-MS/MS)服务将包括蛋白质络合物鉴定，后... 翻译修饰(PTM)定位图，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、总离子流(TIC)、多反应监测(MRM)]。这些 除了来自小鼠和人类的体内组织来源外，还将从细胞系、异种移植组织中进行研究 肿瘤。我们已经开发了代谢组学方面的专业知识，服务将包括极地代谢物 使用选择反应监测(SRM)和极性切换来分析超过300个分子 15分钟。我们将使用稳定状态分析和13C和15N来分析细胞、肿瘤组织和生物液 稳定同位素标记通量实验确定细胞中哪些代谢途径发生了变化 TSC1/2相关通路的缺陷。还将执行非靶向代谢组谱分析，以发现 新的代谢靶点。Core B最近开发了一个基于High的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学识别1000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 还为脂质组学实验开发稳定的同位素通量。除了为项目1-3运行示例外，Core B还开发了一种系列组学技术，利用单个肿瘤、细胞或体液的制备 用于执行三种不同组学(全球磷蛋白质组学/蛋白质组学、代谢组学和 脂质组学)通过分离液-液萃取层。我们还将继续制定-组学战略，以 将模式物种(果蝇)重叠到癌细胞和肿瘤组织以揭示保守的生物相互作用 寻找TSC1/2及相关通路中潜在的生物标记物靶点。