EAGER: Marine biopolymers as tracers of major biogeochemical processes: Using proteomics and antibody-sensor technology

蛋白质组学、生物标志物和生物传感器技术科学的最新进展为理解主要的生物地球化学过程提供了新的途径。该项目将研究海水中模式蛋白“RuBisCO”的物理化学反应性，并将量化北太平洋沿着P线（48°39.0' N，126°40.0' W至50°00' N，145°00' W）的RuBisCO。 该项目将使用两种相互补充和验证的独立方法：免疫传感器和多反应监测（MRM）质谱法。智力优势：化学分析表明，海洋中溶解有机物（DOM）的很大一部分是以蛋白质的形式存在。蛋白质是生物信息的丰富来源，其氨基酸序列提供了与生物体编码DNA的直接联系。DOM中蛋白质的鉴定为理解生物聚合物材料的复杂来源和动力学打开了一扇窗。此外，蛋白质代表了生物体和整个生态系统的催化潜力和反应性。 最重要的是，这些信息还提供了生物和系统发育存在与地球化学之间的直接联系。主要过程特异性蛋白质的分布（例如，RuBisCO到碳固定，固氮酶到固氮）可以用来推断迄今为止一直隐藏的信息。研究人员最近证明了RubisCO在北太平洋的深层分布，并发现这种酶在整个水柱（深度3000米）中的浓度很高，这是高产的赤道和副极地系统的基础，在贫营养的亚热带环流下的浓度很低。这种单一的蛋白质约占1000米深处大部分未鉴定的溶解有机氮库的2%。 RuBisCO的深层分布表明，表层海洋中的水文锋影响了海洋表面以下数千米处最近产生的有机物的分布，而这种酶则追踪了深海环流将深层有机物从输入区域运离的情况。这些发现表明，额外的生物标志物蛋白质的鉴定和定量将提供一个强大的方法来了解相关的生物地球化学途径。虽然这些协议可能具有变革性，但需要加以探讨和验证。 在本研究中，PI将通过了解RuBisCO的物理化学反应性来比较和验证其先前的发现他们将使用两种相互补充和验证的独立和不同的方法来量化新鲜样品中P线的RuBisCO：免疫传感器和多反应监测（MRM）质谱在沿海开放的海洋样带。更广泛的影响：蛋白质组学在水产科学中处于起步阶段，这项研究将有助于推动其发展。将蛋白质组学与生物地球化学相结合将发挥两者的作用。Sorcerer II Global Ocean Sampling（GOS）探险队预测GOS数据库中有600多万种蛋白质，几乎是目前数据库中蛋白质数量的两倍，为已知蛋白质家族增加了巨大的多样性。使用免疫传感器结合S/MRM蛋白质组学将提供关于RuBisCO作为DOM池中的实际生物聚合物的循环的信息。目前的方法使用水解，其仅检测单个氨基酸，但消除了蛋白质的化学历史以及生物学、系统发育存在和生物化学之间的联系。蛋白质免疫传感器将允许在原位跟踪海洋蛋白质，它们的生物合成，转化和降解在未浓缩的海水。此外，了解DOC/DON（溶解有机碳/氮）库中蛋白质的形成，降解和保存将阐明它们在全球碳和氮循环中的作用，并将大大推进我们对海洋地球化学的认识。

Recent advances in proteomics, biomarkers and biosensor technology sciences enable new approaches to understanding major biogeochemical processes. This project will examine the physicochemical reactivity of a model protein "RuBisCO" in seawater, and will quantify RuBisCO along ocean transect Line P (48°39.0' N, 126°40.0' W to 50°00' N, 145°00' W) in the North Pacific Ocean. The project will use two independent methods that complement and validate each other: immune-sensors and multiple-reaction monitoring (MRM) mass spectrometry.Intellectual Merit: Chemical analyses have shown that a significant fraction of dissolved organic matter (DOM) in the ocean is in the form of proteins. Proteins are a rich source of biological information and their amino acid sequence provides a direct link to the coding DNA of an organism. Identification of proteins in DOM opens a window to understanding the complex sources and dynamics of biopolymeric material. Furthermore, proteins represent the catalytic potential and reactivity of an organism and, collectively, of an ecosystem. Most importantly, the information also provides a direct link between biological and phylogenetic presence and biogeochemistry. The distributions of proteins that are specific to major processes (e.g., RuBisCO to carbon fixation, nitrogenase to diazotrophy) can be used to infer information that has been hidden until now. The PIs have recently demonstrated the deep distribution of RubisCO in the North Pacific and discovered this enzyme to be at high concentrations throughout the water column (to depths 3000 m) underlying highly productive equatorial and subpolar systems, and low concentrations under the oligotrophic subtropical gyre. This single protein represents ~2% of the largely unidentified dissolved organic nitrogen pool at depths 1000 m. The deep distribution of RuBisCO shows that hydrographic fronts in the surface ocean affect the distribution of recently-produced organic matter thousands of meters below the ocean surface, and the enzyme traces the transport of deep organic matter by the deep ocean circulation away from regions of input. These findings suggest that the identification and quantification of additional biomarker proteins will provide a powerful approach to understanding the associated biogeochemical pathways. While potentially transformative, the protocols need to be explored and validated. In this study , the PIs will compare and validate their previous findings by understanding the physicochemical reactivity of RuBisCO (and therefore other proteins) in seawater samples in order to fully interpret and exploit the information held in protein distributions; and they will quantify RuBisCO at Line P in fresh samples using two independent and different methods that complement and validate each other: immunosensors and multiple-reaction monitoring (MRM) mass spectrometry in a coastal to open ocean transect.Broader Impacts:Proteomics is in its infancy in the aquatic sciences and this research will help drive its development.Merging the sciences of proteomics with biogeochemistry will leverage both. The Sorcerer II Global Ocean Sampling (GOS) expedition predicted more than six million proteins in the GOS database, almost twice the number of proteins present in the current databases, adding an enormous diversity to the known protein families. Using immunosensors coupled with S/MRM proteomics will provide information on the cycling of RuBisCO as an actual biopolymers in the DOM pool. Current methods use hydrolysis which solely examines single amino acids but erases the chemical history of the proteins and the link between biology, phylogenetic presence, and biogeochemistry. Protein immunosensors will allow in situ tracking of marine proteins, their biosynthesis, transformation, and degradation in unconcentrated seawater. Furthermore, understanding the formation, degradation, and preservation of proteins in the DOC/DON (dissolved organic carbon/nitrogen) pools will elucidate their role in global carbon and nitrogen cycling and will greatly advance our knowledge of marine biogeochemistry.