Current Generation Integrated Instrumentation for H-D Exchange LC-MS

描述(由申请人提供)：本申请申请资金用于购买最先进的多肽酰胺氢-氚交换质谱仪(DXMS)的最新一代仪器，以取代UCSD DXMS资源中目前使用的过时仪器。1.带电子转移解离的Thermo Fisher LTQ Orbitrap Velos质谱仪(ORBXL-0723870)，用于资源中专用的DXMS；2.LEAP技术H/D-X PAL氢-氘交换样品制备和进样平台；3.Waters纳米ACQUITY UPLC系统，用于快速、高分辨率、高灵敏度地分离在LEAP H/D-X PAL上制备的蛋白质水解性消化产物。加州大学圣迭戈分校的DXMS资源为NIH资助的研究人员提供了对最先进的DXMS技术的便捷访问。它的21个主要用户拥有超过30种顶级DXMS重点出版物的显著记录，这些出版物是由资源中所做的工作产生的，他们为依赖资源中所做研究的研究提供的符合S10标准的NIH资金加起来每年的直接成本超过900万美元。虽然该资源一直具有很高的生产力，但连续使用DXMS超过12年的样品制备、色谱和质谱仪(Thermo LCQ Classic和MicroMassQ-TOF I)现在已经过时，必须由支持DXMS的最新一代设备取代。由于我们的能力和科学生产力，对DXMS研究资源的要求正在加速。我们在(样品处理化学)和(数据处理软件)之前和之后采用的DXMS技术的进步，使我们的旧机器在吞吐量和DXMS分析质量方面都成为了限速步骤。虽然任何一种当前一代的质谱计都可以显著提高DXMS研究的吞吐量和质量，但我们的分析和我们顾问的经验表明，Thermo LTQ Orbitrap Velos-ETD是专门用于DXMS的一种卓越且经济高效的仪器。与所需的协同样品制备和LC组件一起，它将使我们能够最好地为我们当前的DXMS用户提供服务，并使我们能够利用DXMS技术的进一步进步，例如ETD介导的氘核子区块化。 公共卫生相关性：在加州大学圣迭戈分校的DXMS资源，我们为许多蛋白质科学家提供了使用技术要求高但功能最强大的基于质谱学的技术(“DXMS”)来精确研究他们感兴趣的蛋白质在执行其生物功能时如何改变形状的能力。不断增长的DXMS使用需求要求资源使用本申请中要求的新一代DXMS仪器更换已经使用了十多年的现已过时的设备。

DESCRIPTION (provided by applicant): This application requests funds to purchase current-generation instrumentation for the performance of state-of- the-art peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS), to replace the obsolete instrumentation presently used in the UCSD DXMS Resource. Integrated sample preparation, liquid chromatography, and mass spectroscopic instrumentation is requested: 1. A Thermo Fisher LTQ Orbitrap Velos mass spectrometer with electron transfer dissociation (ETD) (ORBXL-0723870) for dedicated DXMS use in the Resource; 2. A LEAP Technology H/D-X PAL Hydrogen-Deuterium Exchange sample preparation and injection platform; 3. A Waters nanoACQUITY UPLC System for the performance of rapid, high resolution, and high-sensitivity chromatographic separation of proteolytic digests of deuterated protein samples prepared on the LEAP H/D-X PAL. The UCSD DXMS Resource provides facile access to state-of-the-art DXMS technology to NIH-funded investigators. Its twenty-one Major Users have a remarkable record of more than thirty top-tier DXMS-focused publications resulting from work performed in the Resource, and their combined total S10-eligible NIH funding for research that relies on studies performed in the Resource exceeds $9 M in direct costs per year. While the Resource has been highly productive, the sample preparation, chromatographic, and mass spectroscopic instrumentation (a Thermo LCQ Classic and Micromass Q-TOF I), in continuous DXMS use for more than 12 years, are now obsolete, and must be replaced with current generation DXMS-enabling equipment. Because of our capabilities and scientific productivity, requests to the Resource for DXMS study are accelerating. Advances made in the DXMS technology we employ in front of (sample processing chemistries) and after (data processing software), the mass spectrometers have made our old machines the rate-limiting step in both throughput, and quality of DXMS analysis. While any of a number of current-generation mass spectrometers would provide substantial improved throughput and quality of DXMS study, our analysis, and the experience of our consultants indicates that the Thermo LTQ Orbitrap Velos-ETD is a superior and cost-effective instrument for dedicated DXMS use. Together with the requested synergistic sample preparation and LC components, it will allow us to best provide for our current DXMS users, and position us to exploit further advances in DXMS technology, such as ETD-mediated deuteron sublocalization. PUBLIC HEALTH RELEVANCE: At the UCSD DXMS Resource, we provide many protein scientists with the ability to use a technically demanding, but most powerful mass spectrometry-based technology ("DXMS") to study precisely how the proteins they are interested in change shape as they perform their biologic functions. The growing demand for DXMS use requires that the Resource replace the now obsolete equipment that has been used for more than a decade, with the current generation DXMS instrumentation requested in this application.