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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 质谱和蛋白质组学中心主任负责监督质谱和蛋白质组学中心、人员配备、短期课程培训计划、新用户动手培训计划，监督仪器性能的定期监测，并在PI的协助下领导讨论应考虑和测试的仪器和技术的新方向。将对MALDI、DeCyder、Progenesis、Mascot和SpectraMill的新用户进行培训。 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时间的仪器制造商。 主任将与设备供应商互动，以报告性能问题，安排更换电路板的维修、供应商现场访问和软件升级。他协助PI准备进度报告，赠款更新和新的赠款提案，以改善基础设施，提高研究项目的绩效，但本提案没有预料到。 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。他/她将使用这些技术开展有限的服务工作，以防实验室有动力尝试其样品并使用新的蛋白质组学基础设施和技术研究问题，但没有人员学习该技术。如果最初的实验成功，实验室将被期望提出某人来学习进行和解释实验。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，并将协调系列IEF装置的构建和测试。 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而变化的蛋白质点（蛋白质量，蛋白质活性和/或选定的翻译后修饰）。 目前用于获得用于质谱分析的样品的凝胶内消化和肽提取方法由于消化不良和在威尔斯孔、移液管尖端和管上的损失而倾向于损失较低丰度的样品。多重/多重凝胶扫描优化了随生物刺激而变化的细胞或组织中蛋白质点的位置。 正在建设中的设备将电致变色蛋白质从目标点，流动的样品通过微流体固定胰蛋白酶消化器，并收集样品在多板井MS分析或到Maldi板进行初步测试。我们计划最终将洗脱的肽直接流入Agilent Chip LC的捕获柱，以进行最有效和高回收率的分析。电洗脱路径短（1.0-1.5mm凝胶厚度），固定化胰蛋白酶消化器也很快，每个靶点将花费约10分钟进行消化和消化。在典型的实验中，大量的斑点被分辨，但只有相对少量的蛋白质斑点显示出足够大的强度变化，需要鉴定生物刺激。较强的斑点可以通过使用中的标准斑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小部分。修饰，如磷酸化，将蛋白质转移到2D凝胶上的新位置，因此弱蛋白质点的强度变化通常包含大量的生物信息。在较弱的点发生变化的蛋白质将被构建的电洗脱装置回收。随着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。 HyperScan系统将提供比传统激光扫描仪快得多的扫描（一次扫描所有颜色，每次扫描约400个像素），传统激光扫描仪必须对每个激光激发和发射滤光片组合进行独立扫描。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，并且每个凝胶至少三次颜色扫描，如果每个实验使用多个凝胶，则总扫描时间很长。HyperScan的速度将提高约10倍，灵敏度更高，并且还将允许扩展使用更多的同步多重颜色（扫描时间不会减少），并允许更多的发射光谱重叠，因为更多的Zdye颜色可用。四种颜色在手，五种颜色将很快可用，八种同时的颜色是可行的剂量-响应曲线，时间-响应曲线，同时监测蛋白质量和翻译后修饰，或同时比较更多的样品，大大扩大了样品的吞吐量和信息输出。