Microfluidics for High-Throughput HDX-MS
用于高通量 HDX-MS 的微流控
基本信息
- 批准号:8534211
- 负责人:
- 金额:$ 18.58万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2012
- 资助国家:美国
- 起止时间:2012-08-20 至 2015-05-31
- 项目状态:已结题
- 来源:
- 关键词:AdoptedAmidesAutomationBackBacteriorhodopsinsBasic ScienceBiologicalBiological AssayBiologyClinicalCoupledDataData AnalysesDetergentsDeuteriumDevicesDiagnosticDigestionDissociationElectronsElectrospray IonizationEngineeringEnsureEnvironmental Risk FactorError SourcesGenerationsGoalsHigh Pressure Liquid ChromatographyHigher Order Chromatin StructureHumanHydrogenIntegral Membrane ProteinInterventionJointsLabelLaboratoriesLigandsLogisticsManualsMass Spectrum AnalysisMethodsMicrofluidicsModelingMolecular Sieve ChromatographyMonitorMotivationMyoglobinNatureOrganized by Structure ProteinOutputPathologyPepsin APeptidesPerformancePharmaceutical PreparationsPhasePositioning AttributePreparationProceduresProcessProtein AnalysisProtein ConformationProtein DynamicsProteinsProteomicsProtonsPublishingQuality ControlReactionReproducibilityResearchResearch PersonnelResearch ProposalsSHFM1 geneSafetySamplingSiteSodium ChlorideSolutionsSpeedStructural ProteinSuperoxide DismutaseSystemTechniquesTechnologyTemperatureTestingTherapeuticTimeTranslatingValidationVertebral columnVisionWorkappendagebasecostdata exchangedesigndigitaldrug developmentflexibilityimprovedinsightinstrumentinstrumentationmass spectrometernanolitrenew technologyoperationprogramsprotein complexprotein foldingprotein structurepublic health relevanceresearch study
项目摘要
DESCRIPTION (provided by applicant): The objective of this R21 research proposal is to develop an instrument that can reliably manipulate small amounts of sample to rapidly and automatically perform hydrogen/deuterium exchange (HDX) and be interfaced with current HPLC-ESI-MS systems. We envision this device to be a versatile appendage module to any ESI-MS, already found in many proteomic, biology, and pathology laboratories, and to enable them to perform HDX experiments with minimal human operation. Our long-term goal is to make HDX technology more accessible to a far greater number of researchers who can utilize it to explore protein conformations and dynamics in their studies, at a sensitivity and throughput unmatched by any current instrumentation.
Mass spectrometry-based HDX (HDX-MS) experiments probe protein structures by monitoring the rate and extent of deuterium exchange with backbone amide protons. This approach has proven to be a powerful and versatile protein analysis technique, which can provide valuable insights into protein dynamics in solution such as protein folding, protein-protein complex formation and protein-ligand interactions. Despite these attractive features of HDX technology and its many useful applications, the extensive sample handling necessary to produce the labeled protein provides in itself a source of error, particularly with short incubation times and manual pipetting. Therefore, automation of the labeling procedure would be an advantage. We have developed an automated digital microfluidic droplet generator (DMDG) platform that can generate nanoliter droplets with precisely defined compositions pre-programmed by the user. Our joint team has demonstrated automated multi-step HDX experiments with two model proteins, myoglobin (Mb) and bacteriorhodopsin (bR, an integral membrane protein) using the first-generation DMDG chips coupled with intact protein MS analysis.
Herein, we will develop a DMDG microfluidics instrument, toward minimizing the amount of sample needed, and maximizing the speed of output, under the precise experimental conditions required for HDX experiments. We propose two different chips with (1) parallel channel and (2) variable-volume, single channel designs for HDX testing. Subsequently, we will validate the performance of the second-generation DMDG platform with superoxide-dismutase (SOD1) by site-specific HDX using (1) rapid electron capture/transfer dissociation (ECD/ETD) of intact proteins and (2) pepsin column digestion for peptide validation. Finally, we will explore the possibility of high-throughput HDX-MS using the optimal DMDG chips and multiple micro-size-exclusion chromatography or reverse-phase trap systems. The particularly useful HDX-MS methods, along with the established basic science and engineering programs of our labs, make our mass spectrometry and microfluidic team (J. Whitelegge and C. K.-F. Shen) uniquely positioned to translate new technologies into ongoing scientific discovery at UCLA.
描述(由申请人提供):该R21研究提案的目标是开发一种能够可靠地操作少量样品以快速自动进行氢/氘交换(HDX)并与当前HPLC-ESI-MS系统接口的仪器。我们设想该设备是任何ESI-MS的通用附件模块,已经在许多蛋白质组学,生物学和病理学实验室中发现,并使他们能够以最少的人为操作进行HDX实验。我们的长期目标是使HDX技术更容易为更多的研究人员所使用,他们可以利用它来探索蛋白质构象和动力学,其灵敏度和通量是任何现有仪器所无法比拟的。
基于质谱的HDX(HDX-MS)实验通过监测氘与骨架酰胺质子交换的速率和程度来探测蛋白质结构。这种方法已被证明是一种功能强大且通用的蛋白质分析技术,可以为溶液中的蛋白质动力学提供有价值的见解,例如蛋白质折叠,蛋白质-蛋白质复合物形成和蛋白质-配体相互作用。尽管HDX技术具有这些吸引人的特征及其许多有用的应用,但产生标记蛋白所需的大量样品处理本身就提供了错误的来源,特别是在短孵育时间和手动移液的情况下。因此,标记过程的自动化将是一个优点。我们开发了一种自动数字微流体液滴发生器(DMDG)平台,该平台可以生成纳升液滴,其成分由用户预先编程。我们的联合团队已经证明了使用第一代DMDG芯片结合完整蛋白质MS分析的两种模型蛋白质,肌红蛋白(Mb)和细菌视紫红质(bR,一种整合的膜蛋白)的自动化多步HDX实验。
在此,我们将开发一种DMDG微流体仪器,在HDX实验所需的精确实验条件下,尽可能减少所需的样品量,并最大限度地提高输出速度。我们提出了两种不同的芯片(1)并行通道和(2)可变容量,单通道设计的HDX测试。随后,我们将使用(1)完整蛋白的快速电子捕获/转移解离(ECD/ETD)和(2)用于肽验证的胃蛋白酶柱消化,通过位点特异性HDX验证具有超氧化物歧化酶(SOD 1)的第二代DMDG平台的性能。最后,我们将探索使用最佳DMDG芯片和多个微尺寸排阻色谱或反相捕集系统的高通量HDX-MS的可能性。特别有用的HDX-MS方法,沿着我们实验室建立的基础科学和工程项目,使我们的质谱和微流体团队(J. Whitelegge和C. K.- F. Shen)在加州大学洛杉矶分校将新技术转化为正在进行的科学发现。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Julian P Whitelegge其他文献
Julian P Whitelegge的其他文献
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{{ truncateString('Julian P Whitelegge', 18)}}的其他基金
Organ-specific NRF2-mediated protein signatures of radiation exposure & tissue da
辐射暴露的器官特异性 NRF2 介导的蛋白质特征
- 批准号:
8653935 - 财政年份:2012
- 资助金额:
$ 18.58万 - 项目类别:
Organ-specific NRF2-mediated protein signatures of radiation exposure & tissue da
辐射暴露的器官特异性 NRF2 介导的蛋白质特征
- 批准号:
8469390 - 财政年份:2012
- 资助金额:
$ 18.58万 - 项目类别:
Organ-specific NRF2-mediated protein signatures of radiation exposure & tissue da
辐射暴露的器官特异性 NRF2 介导的蛋白质特征
- 批准号:
8370442 - 财政年份:2012
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Proteomic Approaches to Protein Fatty Acylation
蛋白质脂肪酰化的蛋白质组学方法
- 批准号:
7910658 - 财政年份:2009
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$ 18.58万 - 项目类别:
Subtle Modification of Isotope Ratio Proteomics (SMIRP)
同位素比蛋白质组学的微妙修改 (SMIRP)
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7347308 - 财政年份:2008
- 资助金额:
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Subtle Modification of Isotope Ratio Proteomics (SMIRP)
同位素比蛋白质组学的微妙修改 (SMIRP)
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7770893 - 财政年份:2008
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