MRI: Acquisition of a 600 MHz NMR with cryoprobe for Integration with Existing UPLC-MS-SPE to Enable High Throughput Chemical Annotation in Plant Metabolomics

一个多机构的跨学科研究团队，共同关注植物代谢、生物化学和分子生物学（即代谢组学）的大规模研究，已经联合起来，获得了一台配备cryflowprobe的600 MHz核磁共振（NMR）光谱仪。核磁共振是所有生物体中发现的数千种不同代谢物的化学表征的强大工具。鉴定和化学表征代谢物是了解其生物学、生理学和生态学作用的基本步骤。核磁共振仪器将与现有的超高效液相色谱相结合，结合平行质谱检测和固相萃取（UPLC-MS-SPE），在核磁共振分析之前实现半自动化代谢物纯化和浓缩。该仪器的使用将使俄克拉荷马州东南部集中的生产、尖端和新兴工厂项目得以实现并大大提高。所要求的仪器将由塞缪尔·罗伯茨诺贝尔基金会（Samuel Roberts Noble Foundation）定位并提供支持，该基金会是一家非营利性研究机构，其使命是通过农业的进步来提高人类的水平。占群体的很大比例？我们的代谢组学先前的工作主要集中在基于质谱的方法和模型豆科植物紫花苜蓿的综合功能基因组学。然而，当前和未来的研究范围已经大大扩展到包括其他植物物种，如拟南芥（MCB 0520140 Arabidopsis 2010: MCB 0520283 Arabidopsis 2010），苜蓿，番茄，烟草，啤酒花，棉花，马/树篱苹果，柳枝稷生物燃料，羊茅和相关生物碱产生植物真菌内生菌，以及破坏性真菌植物病原体，如棉花根腐病。随着项目和人员的增加，独特代谢物的数量也在增加。目前，据估计，整个植物界有超过20万种代谢物，其中一半以上仍未被识别。因此，参与小组已经确定了将核磁共振技术纳入其代谢组学和植物生物化学技术基础的关键和迫切需求，以提高他们系统识别、结构表征和比较植物代谢物的能力。目前资助的许多研究都集中在高解剖学分辨率的实验和有限数量的空间上不同的植物器官和细胞类型（如花粉、毛状体、边缘细胞）。因此，生物材料和相应的代谢物丰度很低。因此，配备cryflowprobe的核磁共振与在线质量导向固相萃取相结合，提供了关键的灵敏度增强，这对于在半自动化模式下获得微量代谢物的结构数据是绝对必要的，以提高吞吐量。核磁共振仪器将通过鉴定关键模型和作物物种中重要的新代谢物，促进对植物基本代谢和生物化学的理解。它还将通过增加注释和覆盖深度来增加我们代谢组学项目的生物学背景，这将相应地增强基因发现、基因功能注释和更有效的有益植物性状代谢工程。授权工具资源将用于征聘一名新的长期工作人员。诺布尔基金会将为该仪器和工作人员提供长期支持。资金还将用于招募未来的教师，保留现有的教师，提高植物生物化学和代谢组学参与者的国际竞争力和领导力，并用于利用未来州和联邦对植物生物化学和农业的研究资金。提议的仪器将被热切地纳入所有参与机构现有的和富有成效的教育和培训计划，这些机构为大量不同的博士后、研究生、本科生和高中生提供服务。具体包括：多个动手，深入，为期一周的全国认可的仪器培训课程，如我们的代谢组学和蛋白质组学用户培训计划；本科仪器分析课程；高中科学嘉年华；工具性的“Nut & Volt”利益集团；著名的贵族学者实习计划；以及与俄克拉何马州南部技术中心、阿德莫尔高中AP化学、阿德莫尔高中科学俱乐部和俄克拉何马州向上发展的高中合作项目。

A multi-institutional team of interdisciplinary researchers with a mutual focus upon the large-scale study of plant metabolism, biochemistry, and molecular biology (i.e. metabolomics) have joined together to acquire a 600 MHz nuclear magnetic resonance (NMR) spectrometer equipped with a cryoflowprobe. NMR is a powerful tool for the chemical characterization of the thousands of diverse metabolites found in all living organisms. Identifying and chemically characterizing metabolites is a fundamental step in understanding their biological, physiological, and ecological roles. The NMR instrument will be combined with existing ultra high-performance liquid chromatography coupled to parallel mass spectrometry detection and solid phase extraction (UPLC-MS-SPE) which will enable semi-automated metabolite purification and concentration prior to NMR analysis. Access to this instrument will enable and greatly enhance productive, cutting-edge, and emerging plant programs centralized in southeastern Oklahoma. The requested instrumentation will be located and supported by the Samuel Roberts Noble Foundation, a nonprofit research institute whose mission focuses upon the enhancement of humanity through advancements in agriculture. A significant proportion of the group?s metabolomics prior efforts focused on mass spectrometry based approaches and integrated functional genomics of the model legume Medicago truncatula. However, the scope of current and future studies have substantially expanded to include other plant species such as Arabidopsis thaliana (MCB 0520140 Arabidopsis 2010: MCB 0520283 Arabidopsis 2010), alfalfa, tomato, tobacco, hops, cotton, horse/hedge apple, switchgrass biofuels, fescue and related alkaloid producing plant fungal endophytes, and devastating fungal plant pathogens such as cotton root rot. As the diversity of the group?s programs and staff expands, so does the number of unique metabolites. Currently, it is estimated that there are over 200,000 metabolites throughout the plant kingdom, and over half of these still remain unidentified. Accordingly, the participating groups have identified a critical and urgent need for the acquisition and incorporation of NMR technology into their metabolomics and plant biochemistry technology base to advance their ability to systematically identify, structurally characterize, and comparatively profile plant metabolites. Many of the currently funded studies are focused on high anatomically resolved experiments and limited quantities of spatially distinct plant organs and cell types (i.e. pollen, trichomes, border cells). As a result, biological materials and corresponding metabolites are of very low abundance. Thus, a NMR equipped with a cryoflowprobe and coupled to on-line mass directed solid-phase extraction provides critical sensitivity enhancements that are absolutely necessary for obtaining structural data for minute quantities of metabolites in a semi-automated mode to enhance throughput. The NMR instrumentation will advance the understanding of fundamental plant metabolism and biochemistry through the identification of important novel metabolites in key model and crop species. It will also increase the biological context of our metabolomics programs through increased annotation and depth-of-coverage which will proportionately enhance gene discoveries, functional annotation of genes, and more efficient metabolic engineering of beneficial plant traits. The enabling instrumental resource will be used to recruit one new permanent staff member. Long term support for this instrument and staff will be provided by the Noble Foundation. Funding will also enhance the recruitment of future faculty, retention of current faculty, the international competitiveness and leadership of the participants in plant biochemistry and metabolomics, and used to leverage future state and federal research funding for plant biochemistry and agriculture. The proposed instrumentation will be eagerly incorporated into the existing and productive educational and training programs at all the participating institutes which serve a large number of diverse postdoctoral, graduate, undergraduate, and high school students. These specifically include: multiple hands-on, in-depth, week-long instrumental training courses of national recognition such as our metabolomics and proteomics user training programs; undergraduate instrumental analyses courses; high school Science Carnivals; an instrumental "Nut & Volt" interest group; the prestigious Noble Scholars Intern Program; and high school cooperative programs with the Southern Oklahoma Technology Center, Ardmore High School AP Chemistry, Ardmore High School Science Club, and Oklahoma Upward Bound.