EAGER: Unlocking the clumped isotope signatures of marine nitrate

在许多海洋环境中，氮是限制生命的营养物质。它的可用性决定了海洋生态系统的结构和生产力。为了更好地了解氮的循环，科学家们需要工具来追踪影响自然界氮的过程。过去对硝酸盐氮和氧稳定同位素的研究使我们对全球氮循环有了关键的了解。与此同时，它也凸显了氮循环中许多尚未解决的复杂性。不幸的是，经典的同位素测量方法在捕捉自然界中氮的转化过程方面是有限的。这是因为所得到的同位素特征总是至少三个因素的组合。第一，源化合物的同位素组成。二是过程机制。第三，变换的完备性。在这个探索性研究（EAGER）项目的早期概念资助中，研究人员因此建议探索一种新的分析方法。提出的研究目标是首次测量硝酸盐的双取代或“团块”同位素。块状硝酸盐同位素有可能记录特定的氮转化。这项提议的工作可以解开这些聚集的硝酸盐同位素特征，作为研究海洋氮的新工具。除了技术本身，该项目还将开发用于分析同位素数据的开源软件工具，以支持未来对类似分析物的工作。拟议的项目还将为社区大学生提供参观科罗拉多大学博尔德分校参与实验室的机会。与丹佛地铁STEM少数民族参与联盟的五所社区学院合作，感兴趣的学生将与科罗拉多大学博尔德分校的学生见面，了解正在进行的本科研究、夏季研究机会和四年制大学转学途径。参与的社区学院教育了大量来自弱势群体的学生。为这些学生创造继续获得四年制学位的途径，并在STEM研究和工业领域取得成功的职业生涯，是增加全国STEM学科多样性的关键部分。具体而言，研究人员提出了一种新的电喷雾电离(ESI)-轨道rap质谱法研究海洋硝酸盐同位素的技术方法。这种方法为量化硝酸盐的3个新的同位素维度提供了途径。这些双取代的“团块”硝酸盐同位素（15N18O, 18O18O, 17O18O）以前从未被分析过，它们可以从根本上改变我们对单个氮循环途径如何相互作用以形成海洋生态系统和全球氮循环的理解。因为它们是分子内的同位素维度，这些团块硝酸盐的相对丰度直接反映了形成和破坏它们的过程的动力学和平衡同位素效应。因此，该项目的目标是：1)建立一种ESI-Orbitrap方法，对硝酸盐的所有单一和三种主要的双取代同位素进行可重复的定量分析；2)开发和演示可扩展且易于转移的协议，用于从海洋样品中提取和制备用于ESI-Orbitrap分析的硝酸盐；3)开展海洋样品中硝酸盐块状空间的初步研究。除了对化学海洋学研究的直接影响外，该项目还将向地球化学界介绍新一代完整分子同位素比质谱法，该方法可适用于海洋系统中广泛研究的其他氧离子和代谢物（例如硫酸盐、氨基酸、脂类）。开发硝酸盐作为ESI-Orbitrap同位素分析的模型也将有利于其他科学领域，包括农业研究、陆地生态系统科学和大气化学。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Nitrogen is a limiting nutrient for life in many marine environments. Its availability shapes the structure and productivity of marine ecosystems. To better understand the cycling of nitrogen, scientists need tools to track the processes that affect nitrogen in nature. Past research into the nitrogen and oxygen stable isotopes of nitrate has enabled pivotal insights into the global nitrogen cycle. At the same time, it has highlighted many unresolved complexities in the cycling of nitrogen. Unfortunately, classical isotope measurements are limited in capturing the processes that transform nitrogen in nature. This is because the resulting isotope signatures are always a combination of at least three factors. First, the isotopic composition of the source compound. Second, the mechanism of the process. And third, the completeness of the transformation. In this EArly-concept Grant for Exploratory Research (EAGER) project, the investigators thus propose to explore a new analytical approach. The goal of the proposed research is to measure the doubly-substituted or “clumped” isotopologs of nitrate for the first time. Clumped nitrate isotopes have the potential to record specific nitrogen transformations. The proposed work could unlock these clumped nitrate isotope signatures as a new tool to study nitrogen in the oceans. In addition to the technology itself, this project will develop open-source software tools for the analysis of isotopolog data to support future work on similar analytes of interest. The proposed project will also create an opportunity for community college students to visit participating laboratories at CU Boulder. In collaboration with the five community colleges of the Denver Metro STEM Alliance for Minority Participation, interested students will meet current CU Boulder students, learn about ongoing undergraduate research, summer research opportunities and 4-year college transfer pathways. The participating community colleges educate a large number of students from underrepresented backgrounds. Creating pathways for these students to continue on to a 4-year degree and a successful career in STEM research and industry fields is a critical piece of the puzzle for increasing diversity in STEM disciplines nationwide.Specifically, the investigators propose to develop a new technological approach to studying the isotopes of marine nitrate by electrospray ionization (ESI)-Orbitrap mass spectrometry. This approach provides a path to quantifying up to 3 new isotopic dimensions of nitrate. These doubly substituted, “clumped” nitrate isotopologs (15N18O, 18O18O, 17O18O) have never before been analytically accessible and could fundamentally transform our understanding of how individual nitrogen cycling pathways interact to shape ocean ecosystems and the global nitrogen cycle. Because they are intramolecular isotopic dimensions, the relative abundances of these clumped nitrate species directly reflect kinetic and equilibrium isotope effects of the processes that form and destroy them. The objectives of this project are thus to 1) establish an ESI-Orbitrap methodology for the reproducible quantification of all singly and the three major doubly substituted isotopologs of nitrate; 2) develop and demonstrate scalable and easily transferable protocols for the extraction and preparation of nitrate from marine samples for ESI-Orbitrap analysis; 3) initiate a pilot study of the clumped nitrate space in marine samples. Beyond its immediate impact on research in chemical oceanography, this project will introduce the geochemistry community to a new generation of intact molecule isotope-ratio mass spectrometry that can be adapted to other oxyanions and metabolites widely studied in marine systems (e.g., sulfate, amino acids, lipids). Developing nitrate as a model for ESI-Orbitrap isotope analytics also will benefit other fields of science including agricultural research, terrestrial ecosystems science, and atmospheric chemistry.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.