Microfluidics for High-Throughput HDX-MS

描述(由申请人提供)：本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/氢交换(HDX)，并与当前的高效液相-电喷雾串联质谱仪系统接口。我们设想该设备将成为任何ESI-MS的通用附件模块，这些模块已经在许多蛋白质组、生物学和病理学实验室中找到，并使他们能够以最少的人工操作进行HDX实验。我们的长期目标是让更多的研究人员更容易获得HDX技术，他们可以利用HDX技术在他们的研究中探索蛋白质构象和动力学，其灵敏度和吞吐量是目前任何仪器都无法比拟的。 基于质谱学的HDX(HDX-MS)实验通过监测与主链酰胺质子交换重氢的速度和程度来探测蛋白质结构。这种方法已被证明是一种强大而通用的蛋白质分析技术，它可以为蛋白质在溶液中的动态变化提供有价值的见解，如蛋白质折叠、蛋白质-蛋白质复合体的形成和蛋白质-配体的相互作用。尽管HDX技术具有这些吸引人的特点及其许多有用的应用，但生产标记蛋白质所需的大量样品处理本身就提供了一个误差来源，特别是孵育时间短和手动移液。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数字微流控液滴发生器(DMDG)平台，可以生成用户预先编程的精确定义成分的纳升液滴。我们的联合团队使用第一代DMDG芯片和完整的蛋白质MS分析，展示了两种模型蛋白质--肌红蛋白(Mb)和细菌视紫红质(BR，一种完整的膜蛋白质)--的自动化多步HDX实验。 在这里，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，实现所需样品量的最小化和输出速度的最大化。针对HDX测试，我们提出了两种不同的芯片设计：(1)并行通道设计和(2)可变体积单通道设计。随后，我们将通过(1)完整蛋白质的快速电子捕获/转移解离(ECD/ETD)和(2)用于肽验证的胃蛋白柱消化来验证具有超氧化物歧化酶(SOD1)的第二代DMDG平台的性能。最后，我们将探索使用最优的DMDG芯片和多个微尺寸排除层析或反相捕集系统进行高通量HDX-MS的可能性。特别有用的HDX-MS方法，以及我们实验室已建立的基础科学和工程计划，使我们的质谱学和微流控团队(J.Whitelege和C.K.-F.Shenin)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。

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.