Developing position-specific amino acid carbon isotope analysis as a tool for geobiology

地球生物学的一个长期目标是发展重建和理解生物体在其环境中过去和现在的代谢活动的方法。在这里，研究人员通过开发新的方法来精确测量氨基酸不同原子位置中碳的重稳定同位素（C-13）的丰度，向这一目标迈进了一步。他的预测是，这些同位素丰度的模式将作为不同代谢途径的“指纹”，当从环境蛋白质中测量氨基酸时，将允许从该环境中推断生物体的代谢活动。他将采用一种相对较新的质谱法（Orbitrap MS），非常精确地测量几个分子片段的C-13/C-12比率。这些重叠的碎片可以在数学上重新组合成每个原子位置的同位素丰度模型。作为新测量的测试案例，他将检验以下三个假设。1)所有氨基酸中同位素丰度的模式将根据宿主生物体如何固定碳而有所不同，无论是通过卡尔文循环（像所有植物一样）还是通过细菌使用的许多替代途径。2)氨基酸谷氨酸和脯氨酸的同位素模式将反映生物体的生长效率，因为这两种化合物之间存在特定的代谢联系。3)丝氨酸和丙氨酸中的同位素模式将反映被称为光呼吸的过程的程度，该过程发生在植物中，随着温度的升高，有效地逆转了光合作用的工作。在单个原子位置水平上测量同位素组成将提供前所未有的能力来重建新陈代谢，环境过程和其他地质生物学现象。该研究将采用Eiler等人（2017）描述的现有质谱技术，其中通过轨道阱质谱法以高质量分辨率（高达500，000 M/Delta M）测量特定分析物的分子离子和各种碎片离子的C-13/C-12比率，以区分同量异位素。分析物的每个原子位置对每个碎片离子的相对贡献（“贡献矩阵”）必须通过对标记化合物的研究来确定，然后可以从碎片中重建完整的位置特异性Δ C-13值。该项目将开发测量方案，包括贡献矩阵，用于六种氨基酸中的位置特异性Δ C-13：丙氨酸，丝氨酸，谷氨酸，脯氨酸，天冬氨酸和蛋氨酸。这些共同跨越了所有主要的碳骨架家族，这些家族是由氨基酸采样的。研究人员将使用这些新工具来探索三个重点假设：1）所有氨基酸（包括Ala，Glu和Met）中的位置特异性Δ 13-C将反映亲本生物体的碳固定途径。2)Glu和Pro中C-2与C-3的位置特异性C-13分馏应反映α-酮戊二酸脱羧与转氨的程度，他预测这与生长效率成比例。3)他预测，C-13分馏在C-2在丝氨酸与丙氨酸应与光呼吸的程度covariate，因为氧化RuBP是通过丝氨酸pathway.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

A longstanding goal in geobiology has been to develop ways of reconstructing and understanding the metabolic activities of organisms, both past and present, in their environment. Here the investigator takes a step toward that goal by developing new methods to precisely measure the abundance of the heavy stable isotope of carbon (C-13) in different atomic positions of amino acids. His prediction is that these patterns of isotope abundance will serve as a 'fingerprint' of different metabolic pathways, and when measured in amino acids from environmental proteins will allow to infer the metabolic activities of organisms from that environment. He will employ a relatively new mass spectrometry method (Orbitrap MS) that measures the C-13/C-12 ratios of several molecular fragments very precisely. These overlapping fragments can then be mathematically recombined into a model of isotope abundance at each atomic position. As test cases for the new measurements, he will examine the following three hypotheses. 1) The pattern of isotope abundance in all amino acids will differ depending on how the host organism fixes carbon, whether by the Calvin Cycle (like all plants) or via a number of alternative pathways used by bacteria. 2) The isotope pattern in the amino acids glutamate and proline will reflect the growth efficiency of the organism, because of the specific metabolic connection between these two compounds. 3) The isotope pattern in serine and alanine will reflect the extent of a process known as photorespiration, which occurs in plants as temperatures rise and effectively reverses the work of photosynthesis.The overarching goal of this proposal is to develop position-specific delta C-13 measurements in amino acids as a tool for geobiologic research. Measurements of isotopic composition at the level of individual atomic positions will provide unprecedented ability to reconstruct metabolism, environmental processes, and other geobiologic phenomena. The investigation will employ existing mass spectrometric techniques described by Eiler et al (2017) in which the C-13/C-12 ratios of the molecular ion and various fragment ions of a particular analyte are measured via Orbitrap mass spectrometry at high mass resolution (up to 500,000 M/Delta M) to distinguish isobars. The relative contributions of each atomic position of the analyte to each fragment ion (the 'contribution matrix') must be determined through studies of labeled compounds, and then the complete position-specific delta C-13 values can be reconstructed from the fragments. This project will develop measurement protocols, including contribution matrices, for position-specific delta C-13 in six amino acids: alanine, serine, glutamate, proline, aspartate, and methionine. These collectively span all of the major families of carbon skeletons that are sampled by amino acids. The investigator will use these new tools to explore three focused hypotheses: 1) Position-specific delta 13-C in all amino acids, include Ala, Glu, and Met, will reflect the carbon-fixation pathway of the parent organism. 2) Position-specific C-13 fractionation of C-2 versus C-3 in Glu and Pro should reflect the extent to which alpha-ketoglutarate is decarboxylated versus transaminated, which he predicts is proportionate to growth efficiency. 3) He predicts that C-13 fractionation at C-2 in Ser versus Ala should covary with the extent of photorespiration, since oxidized RuBP is recycled via the serine pathway.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.