MASS SPECTROMETRY

质谱

• 批准号: 8167556
• 负责人: BRIAN P BOTHNER
• 金额: $ 44.68万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167556
• 关键词: Applications Grants; Biological; Cells; Centers of Research Excellence; Color; Computer Retrieval of Information on Scientific Projects Database; Computer software; Devices; Digestion; Dose; Equipment; Funding; Gel; Grant; Hand; Human Resources; Institution; Laboratories; Lasers; Learning; Location; Mass Spectrum Analysis; Measurement; Microfluidics; Minor; Modification; Monitor; Output; Peptides; Performance; Phosphorylation; Positioning Attribute; Post-Translational Protein Processing; Process; Progress Reports; Proteins; Proteomics; Protocols documentation; Quality Control; Reaction Time; Recovery; Reporting; Research; Research Infrastructure; Research Personnel; Research Project Grants; Resources; Robot; Running; Sampling; Scanning; Services; Site Visit; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; System; Technology; Testing; Thick; Time; Tissues; Training; Training Programs; Trypsin; Tube; United States National Institutes of Health; Universities; Vendor; Work; instrument; instrumentation; multiplex detection; numb protein; operation; pointed protein; repaired; research study; response; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Director of the Mass Spectrometry and Proteomics Core oversees the Mass Spectrometry and Proteomics Core, the staffing, the Short Course Training Program, New User Hands-On Training Program, oversee regular monitoring of instrument performance, and leads, with the assistance of the PI, discussions of new directions in instrumentation and technology that should be considered and tested. Training of new users in MALDI, DeCyder, Progenesis, Mascot, and SpectraMill will be carried out. Staff will include two full time technicians, one supported by COBRE and the second by University and Facility funds, and a 3/4 time instrument maker. The Director will interact with the equipment vendors to report performance issues, arrange for board-swap repairs to be made, vendor site visits, and software upgrades. He assists the PI in preparing progress reports, grant renewals and new grant proposals for infrastructure enhancements that could enhance research project performance, but were not anticipated by the present proposal. The Research Technician will be responsible for training new users in 2D gel technology, in protein digestion and the operation of the nanoLC/ChipLC/lon Trap LC/MS/MS. He/she will carry out limited service work using these technologies, in cases were laboratories are motivated to try their samples and their research problems with the new proteomics infrastructure and technology, but do not have the personnel available to learn the technology. If initial experiments are successful the laboratories will be expected to put someone forward to learn to carry out and interpret the experiments. He/she will also be responsible for running and recording frequent quality control tests. Instrument Maker (75% time) will build, test and maintain a targeted gel spot electro-elution device mounted on a gel spot picking robot and will coordinate the building and testing of a serial IEF apparatus. The new Zdye proteomics multiplex detection system can pin-point protein spots that change with biological stimulation (for protein amount, protein activity, and/or in selected post-translational modifications). The in-gel digestion and peptide extraction approach currently used to obtain samples for mass spectrometric analysis tends to lose lower abundance samples, due to poor digestion and losses on wells, pipet tips and tubes. The multicolor/multiplex gel scanning optimizes the location of protein spots from cells or tissues that change with biological stimulation. The device under construction will electro-elute proteins from the targeted spots, flow the sample through a microfluidic immobilized trypsin digester, and collect the sample in a multiplate well for MS analysis or onto a Maldi plate for initial testing. We plan to ultimately flow the eluted peptides directly into the trapping column of the Agilent Chip LC, for the most efficient and high-recovery analysis. The electro elution path is short (the 1.0-1.5mm gel thickness) and the immobilized trypsin digester is also fast and each targeted spot will take about 10 min to elute and digest. A vast number of spots are resolved in typical experiments but only a relatively small number of protein spots show sufficiently large changes in intensity, with biological stimulation to require identification. The stronger spots can be removed and processed by the standard spot-cutting robot protocol in use or by electro-elution and microfluidic digestion. Many of the "control" proteins in cells, such as transcription factors, are present in low copy number and their active forms are typically a minor fraction that is phosphorylated. Modifications, such as phosphorylation, shift the proteins to new positions on 2D gels and so changes in the intensities of weak protein spots often contain a great deal of biological information. The proteins that change in the weaker spots will be recovered by the electro elution device to be constructed. The stronger spots that change with biological stimulation will be recovered by the standard spot cutting robot system in use now. The HyperScan system will provide much faster scanning (one pass for all colors and measurement of about 400 pixels per pass) than conventional laser scanners that must carry out independent scans for each single laser excitation and emission filter combination. The current best laser gel scanner is the GE Typhoon Trio (which we have at MSU) that takes 20 min/per scan color on large format gel, and a minimum three color scans per gel, for a long total scan time if multiple gels are used per experiment. The HyperScan will be about 10x faster, be more sensitive, and will also allow expansion to use many more simultaneous multiplex colors (with no decrease in scan time) and allow much more overlap of emisson spectra, as more Zdye colors become available. Four colors are in hand, five colors will soon be available, and eight simultaneous colors are feasible for dose-response curves, time-response curves, simultaneous monitoring protein amounts and post-translational modifications, or comparison of many more samples simultaneously for greatly expanded sample throughput and information output.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 质谱和蛋白质组学核心主任负责监督质谱和蛋白质组学核心、人员配置、短期课程培训计划、新用户实践培训计划，监督仪器性能的定期监测，并在 PI 的协助下领导对应考虑和测试的仪器和技术新方向的讨论。将进行 MALDI、DeCyder、Progenesis、Mascot 和 SpectraMill 的新用户培训。 工作人员将包括两名全职技术人员，一名由 COBRE 支持，另一名由大学和设施基金支持，以及一名 3/4 时间仪器制造商。 总监将与设备供应商互动，报告性能问题，安排换板维修、供应商现场访问和软件升级。他协助 PI 准备进度报告、拨款续签和新的拨款提案，以增强基础设施，从而提高研究项目的绩效，但目前的提案并未预料到。 研究技术员将负责对新用户进行 2D 凝胶技术、蛋白质消化和 nanoLC/ChipLC/lon Trap LC/MS/MS 操作方面的培训。如果实验室有动力尝试新的蛋白质组学基础设施和技术的样品和研究问题，但没有人员可以学习该技术，他/她将使用这些技术开展有限的服务工作。如果最初的实验成功，实验室将需要派人来学习如何进行和解释实验。他/她还将负责运行和记录频繁的质量控制测试。 仪器制造商（75% 时间）将构建、测试和维护安装在凝胶点拾取机器人上的目标凝胶点电洗脱装置，并将协调串行 IEF 设备的构建和测试。 新的 Zdye 蛋白质组学多重检测系统可以精确定位随生物刺激（蛋白质含量、蛋白质活性和/或选定的翻译后修饰）而变化的蛋白质点。 目前用于获取质谱分析样品的凝胶内消化和肽提取方法往往会丢失丰度较低的样品，因为消化不良以及孔、移液器吸头和试管上的损失。多色/多重凝胶扫描优化了细胞或组织中随生物刺激而变化的蛋白质点的位置。 正在构建的设备将从目标点电洗脱蛋白质，使样品流过微流体固定胰蛋白酶消化器，并将样品收集在多孔板孔中进行 MS 分析或收集到 Maldi 板上进行初始测试。我们计划最终将洗脱的肽直接流入安捷伦芯片液相色谱的捕获柱，以实现最高效和高回收率的分析。电洗脱路径短（凝胶厚度1.0-1.5mm），固定化胰蛋白酶消化速度也快，每个目标点大约需要10分钟来洗脱和消化。在典型的实验中，大量的斑点被解析，但只有相对少量的蛋白质斑点显示出足够大的强度变化，需要进行生物刺激来识别。较强的斑点可以通过使用的标准点切割机器人方案或通过电洗脱和微流体消化来去除和处理。 细胞中的许多“对照”蛋白质（例如转录因子）以低拷贝数存在，并且它们的活性形式通常是磷酸化的一小部分。磷酸化等修饰会将蛋白质转移到 2D 凝胶上的新位置，因此蛋白质弱点强度的变化通常包含大量生物信息。在较弱部位发生变化的蛋白质将被所构建的电洗脱装置回收。随生物刺激而变化的较强斑点将由现在使用的标准斑点切割机器人系统恢复。 与必须对每个激光激发和发射滤光片组合进行独立扫描的传统激光扫描仪相比，HyperScan 系统将提供更快的扫描速度（一次扫描所有颜色，每次测量约 400 个像素）。目前最好的激光凝胶扫描仪是 GE Typhoon Trio（我们在密歇根州立大学拥有），在大幅面凝胶上每次扫描颜色需要 20 分钟，并且每个凝胶至少进行三种颜色扫描，如果每次实验使用多个凝胶，则总扫描时间较长。随着更多 Zdye 颜色的出现，HyperScan 的速度将提高约 10 倍，更加灵敏，并且还允许扩展以使用更多同时多重颜色（扫描时间不会减少），并允许发射光谱更多的重叠。四种颜色已在手，五种颜色即将推出，八种同步颜色可用于剂量反应曲线、时间反应曲线、同时监测蛋白质含量和翻译后修饰，或同时比较更多样品，以大大扩展样品通量和信息输出。