Synapt G2-S System with HXMS Automation

具有 HXMS 自动化功能的 Synapt G2-S 系统

• 批准号: 8447332
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 59.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8447332
• 关键词: Automation; Binding; Biological Factors; California; Chloroplasts; Chromatography; Circadian Rhythms; Computer software; Data; Data Analyses; Deuterium; Digestion; Drug Delivery Systems; Engineering; Funding; Future; Housing; Hydrogen; I-kappa B Proteins; Injection of therapeutic agent; Interleukins; Ions; Manuals; Maps; Mass Spectrum Analysis; Measures; Micelles; Optics; Pepsin A; Phosphotransferases; Preparation; Proteins; Proteomics; Research; Research Personnel; Sampling; Signal Recognition Particle; Spectrometry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Tobacco; United States National Institutes of Health; Universities; Water; aristolochene synthase; base; inhibitor/antagonist; instrument; ion source; mass spectrometer; novel; receptor; research study; terpene synthase

DESCRIPTION (provided by applicant): The Biomolecular and Proteomics Mass Spectrometry Facility (BPMSF) at the University of California San Diego wishes to acquire a Synapt G2-S System with HXMS Automation. The PI of this project was the first to show that hydrogen/deuterium exchange (HX) could be measured by MALDI mass spectrometry. Her lab was one of the first to use this experimental approach for mapping macromolecular interfaces. Since 1998, the MALDI-TOF mass spectrometer in the BPMSF has been continuously used for HDX experiments, mainly performed by biochemists who were not experts in mass spectrometry. The Synapt G2-S System with HXMS Automation is the first fully-automated instrument that can replace the MALDI-TOF for use by non-mass spectrometry experts. The Waters instrument also provides many improvements in sensitivity, automation, and data analysis compared with the old manual MALDI-TOF approach. The integrated chromatographic system is based on the Waters nanoACQUITY platform and contains a Peltier-cooled module that houses the injection and switching valves, online pepsin digestion and C-18 separation columns. This chromatographic system is fully integrated with the qTOF mass spectrometer which combines dual-T-wave, off-axis ion transfer optics to transfer ions from the ion source to the quadrupole MS analyzer with the highest possible efficiency. A suite of software coordinates the sample preparation, chromatography, and mass spectrometry and provides rapid and automated data raw viewing, analysis and quantitation with the DynamX software. The Major Users are all researchers who have in the past, or will in the future attempt HDX experiments in the BPMSF. Their projects encompass the gamut of NIH-funded research. The Synapt G2-S System with HXMS Automation will enable new and important discoveries not possible with the outdated MALDI-TOF approach: Komives will measure the "foldedness" of inhibitors of NF- kappa B proteins and changes in their folded state upon binding to their targets; Ghosh will analyze conformational changes in the kinase (IKK) that phosphorylates these inhibitors targeting them for degradation; Noel will analyze conformational changes in Tobacco 5-epi-aristolochene synthase and other terpene synthases which they are engineering to make novel natural products; Gianneschi will probe the macrostructure of proteinaceous micelles for drug delivery; Jennings will analyze the conformational changes in interleukins and kinases; Shan will map the substrate- and receptor- interactions in the chloroplast Signal Recognition Particle; and Kay will screen modulators of circadian rhythm-control proteins.

描述（由申请人提供）： 加州大学圣地亚哥分校的生物分子和蛋白质组学质谱设施 (BPMSF) 希望获得具有 HXMS 自动化功能的 Synapt G2-S 系统。该项目的PI首次证明可以通过MALDI质谱法测量氢/氘交换（HX）。她的实验室是最早使用这种实验方法来绘制大分子界面的实验室之一。自1998年以来，BPMSF的MALDI-TOF质谱仪一直用于HDX实验，主要由非质谱专家的生物化学家进行。具有 HXMS 自动化功能的 Synapt G2-S 系统是首款可以替代 MALDI-TOF 供非质谱专家使用的全自动仪器。与旧的手动 MALDI-TOF 方法相比，沃特世仪器还在灵敏度、自动化和数据分析方面提供了许多改进。集成色谱系统基于沃特世 nanoACQUITY 平台，包含珀耳帖冷却模块，该模块装有进样阀和切换阀、在线胃蛋白酶消化和 C-18 分离柱。该色谱系统与 qTOF 质谱仪完全集成，后者结合了双 T 波、离轴离子传输光学器件，以尽可能高的效率将离子从离子源传输到四极杆 MS 分析仪。一套软件协调样品制备、色谱分析和质谱分析，并通过 DynamX 软件提供快速、自动化的数据原始查看、分析和定量。主要用户是所有过去或将来在 BPMSF 中尝试 HDX 实验的研究人员。他们的项目涵盖了 NIH 资助的所有研究。具有 HXMS 自动化功能的 Synapt G2-S 系统将实现过时的 MALDI-TOF 方法无法实现的新的重要发现：Komives 将测量 NF-kappa B 蛋白抑制剂的“折叠度”以及其折叠状态在与目标结合后的变化； Ghosh 将分析激酶（IKK）的构象变化，该激酶使这些抑制剂磷酸化，从而靶向它们进行降解； Noel 将分析烟草 5-表马兜铃烯合酶和其他萜烯合酶的构象变化，他们正在改造这些酶来制造新型天然产物； Gianneschi 将探索用于药物输送的蛋白质胶束的宏观结构；詹宁斯将分析白细胞介素和激酶的构象变化； Shan 将绘制叶绿体信号识别颗粒中底物和受体相互作用的图谱；凯将筛选昼夜节律控制蛋白的调节剂。

项目成果

The folding unit of phosphofructokinase-2 as defined by the biophysical properties of a monomeric mutant.

由单体突变体的生物物理特性定义的磷酸果糖激酶-2 的折叠单位。

• DOI: 10.1016/j.bpj.2015.04.001
• 发表时间: 2015
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Ramírez-Sarmiento,CésarA;Baez,Mauricio;Zamora,RicardoA;Balasubramaniam,Deepa;Babul,Jorge;Komives,ElizabethA;Guixé,Victoria
• 通讯作者: Guixé,Victoria