Microfluidics for High-Throughput HDX-MS

用于高通量 HDX-MS 的微流控

• 批准号: 8353339
• 负责人: Julian P Whitelegge
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/8353339
• 关键词: Adopted; Amides; Automation; Back; Bacteriorhodopsins; Basic Science; Biological; Biological Assay; Biology; Clinical; Coupled; Data; Data Analyses; Detergents; Deuterium; Devices; Diagnostic; Digestion; Dissociation; Electrons; Electrospray Ionization; Engineering; Ensure; Environmental Risk Factor; Error Sources; Generations; Goals; High Pressure Liquid Chromatography; Higher Order Chromatin Structure; Human; Hydrogen; Integral Membrane Protein; Intervention; Joints; Label; Laboratories; Ligands; Logistics; Manuals; Mass Spectrum Analysis; Methods; Microfluidics; Modeling; Molecular Sieve Chromatography; Monitor; Motivation; Myoglobin; Nature; Organized by Structure Protein; Output; Pathology; Pepsin A; Peptides; Performance; Pharmaceutical Preparations; Phase; Positioning Attribute; Preparation; Procedures; Process; Protein Analysis; Protein Conformation; Protein Dynamics; Proteins; Proteomics; Protons; Publishing; Quality Control; Reaction; Reproducibility; Research; Research Personnel; Research Proposals; SHFM1 gene; Safety; Sampling; Site; Sodium Chloride; Solutions; Speed; Structural Protein; Superoxide Dismutase; System; Techniques; Technology; Temperature; Testing; Therapeutic; Time; Translating; Validation; Vertebral column; Vision; Work; appendage; base; cost; data exchange; design; digital; drug development; flexibility; improved; insight; instrument; instrumentation; mass spectrometer; nanolitre; new technology; operation; programs; protein complex; protein folding; protein structure; research study

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. PUBLIC HEALTH RELEVANCE (provided by applicant): The goal of this proposal is to develop a microfluidic hydrogen/deuterium exchange (HDX) instrument, which is comprehensive, flexible, rapid, and robust, to automate the sample preparation process for interfacing with mass spectrometry for HDX analyses. We envision this device to be a versatile appendage module to any electrospray-ionization mass spectrometer, which already exist in many research and clinical laboratories, 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, ultimately in a high-throughput format.

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的仪器，以快速、自动地执行氢/氘交换 (HDX)，并与当前的 HPLC-ESI-MS 系统连接。我们设想该设备将成为任何 ESI-MS 的多功能附属模块（已在许多蛋白质组学、生物学和病理学实验室中使用），并使它们能够以最少的人工操作执行 HDX 实验。我们的长期目标是让更多的研究人员更容易使用 HDX 技术，他们可以利用该技术在研究中探索蛋白质构象和动力学，其灵敏度和通量是任何当前仪器都无法比拟的。 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强大且多功能的蛋白质分析技术，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性及其许多有用的应用，但生产标记蛋白所需的大量样品处理本身就提供了错误来源，特别是在短孵育时间和手动移液的情况下。因此，标签程序的自动化将是一个优势。我们开发了一种自动化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精确定义。我们的联合团队使用第一代 DMDG 芯片与完整蛋白质 MS 分析相结合，展示了两种模型蛋白质：肌红蛋白 (Mb) 和细菌视紫红质（bR，一种完整膜蛋白）的自动化多步骤 HDX 实验。 在此，我们将开发一种 DMDG 微流控仪器，在 HDX 实验所需的精确实验条件下，最大限度地减少所需的样品量，并最大限度地提高输出速度。我们提出两种不同的芯片，具有 (1) 并行通道和 (2) 可变容量、单通道设计，用于 HDX 测试。随后，我们将通过位点特异性 HDX 验证具有超氧化物歧化酶 (SOD1) 的第二代 DMDG 平台的性能，使用 (1) 完整蛋白质的快速电子捕获/转移解离 (ECD/ETD) 和 (2) 胃蛋白酶柱消化进行肽验证。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获系统进行高通量 HDX-MS 的可能性。特别有用的 HDX-MS 方法，加上我们实验室已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）处于独特的地位，能够将新技术转化为加州大学洛杉矶分校正在进行的科学发现。 公共健康相关性（由申请人提供）：本提案的目标是开发一种全面、灵活、快速且稳健的微流控氢/氘交换（HDX）仪器，以自动化样品制备过程，以便与质谱分析进行 HDX 分析。我们设想该设备将成为任何电喷雾电离质谱仪的多功能附属模块，该模块已存在于许多研究和临床实验室中，使它们能够以最少的人工操作执行 HDX 实验。我们的长期目标是让更多的研究人员更容易使用 HDX 技术，他们可以利用该技术在研究中探索蛋白质构象和动力学，最终以高通量的形式。