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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 质谱学和蛋白质组学核心主管监督质谱学和蛋白质组学核心、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。将对新用户进行MALDI、DeCyder、Progensis、Mascot和spectraMill方面的培训。 员工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。主任将与设备供应商互动，以报告性能问题、安排进行板换维修、供应商现场访问和软件升级。他协助私人投资编写进度报告、赠款续期和新的赠款提案，以加强基础设施，从而提高研究项目的绩效，但本提案没有预料到这一点。 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/她将使用这些技术进行有限的服务工作，在实验室被激励尝试他们的样品以及他们对新的蛋白质组学基础设施和技术的研究问题的情况下，他/她将没有人员来学习该技术。如果最初的实验成功，预计实验室将提出一个人来学习执行和解释实验。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%的时间)将建造、测试和维护安装在凝胶斑点拾取机器人上的目标凝胶点电洗脱装置，并将协调建造和测试一系列IEF装置。 新的扎德蛋白质组学多重检测系统可以定位随着生物刺激(蛋白质数量、蛋白质活性和/或选定的翻译后修饰)而变化的蛋白质斑点。目前用于获得质谱分析样品的凝胶内消化和多肽提取方法，由于消化不良以及井、吸管顶端和试管的损失，往往会损失丰度较低的样品。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斑点的位置。正在建设中的设备将从目标位置电洗脱蛋白质，通过微流体固定化胰酶消化器流动样本，并将样本收集在多板井中进行MS分析或在MALDI板上进行初步测试。我们计划最终将洗脱的多肽直接流入安捷伦芯片LC的捕捉柱，以进行最有效和高回收率的分析。电洗脱路径短(凝胶厚度1.0-1.5 mm)，固定化胰酶消化池速度快，每个靶点需要10分钟左右的洗脱消化时间。在典型的实验中，大量的斑点被分辨出来，但只有相对少数的蛋白质斑点显示出足够大的强度变化，需要生物刺激才能识别。更强的斑点可以通过正在使用的标准斑点切割机器人方案或通过电洗脱和微流控消化来去除和处理。 细胞中的许多“控制”蛋白，如转录因子，以低拷贝数存在，它们的活性形式通常是一小部分被磷酸化的部分。修饰，如磷酸化，使蛋白质在2D凝胶上移动到新的位置，因此蛋白质薄弱点强度的变化通常包含大量的生物信息。在弱点发生变化的蛋白质将通过待构建的电洗脱装置进行回收。随着生物刺激而变化的较强的斑点将通过目前使用的标准斑点切割机器人系统来恢复。 HyperScan系统将提供比传统激光扫描仪快得多的扫描速度(所有颜色一次扫描，每次测量约400像素)，传统激光扫描仪必须对每个单一的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 Typhon Trio(我们在密歇根大学拥有)，它在大幅面凝胶上每扫描一种颜色需要20分钟，每种凝胶至少三次彩色扫描，如果每次实验使用多个凝胶，则总扫描时间较长。HyperScan将更快约10倍，更灵敏，还将允许扩展使用更多的同时多路复用颜色(扫描时间不会减少)，并允许更多的发射光谱重叠，因为有更多的Zdy颜色可用。有四种颜色在手，五种颜色即将推出，八种同时颜色可用于剂量-反应曲线、时间-反应曲线、同时监测蛋白质数量和翻译后修饰，或者同时比较更多的样本，以大大扩大样本吞吐量和信息输出。