State Key Laboratory of Medical Proteomics, CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2024 Jul 23;96(29):12030-12039. doi: 10.1021/acs.analchem.4c02079. Epub 2024 Jul 13.
Marine microbes drive pivotal transformations in planetary-scale elemental cycles and have crucial impacts on global biogeochemical processes. Metaproteomics is a powerful tool for assessing the metabolic diversity and function of marine microbes. However, hundreds of liters of seawater are required for normal metaproteomic analysis due to the sparsity of microbial populations in seawater, which poses a substantial challenge to the widespread application of marine metaproteomics, particularly for deep seawater. Herein, a sensitive marine metaproteomics workflow, named sensitive marine metaproteome analysis (SMMP), was developed by integrating polycarbonate filter-assisted microbial enrichment, solid-phase alkylation-based anti-interference sample preparation, and narrow-bore nanoLC column for trace peptide separation and characterization. The method provided more than 8500 proteins from 1 L of bathypelagic seawater samples, which covered diverse microorganisms and crucial functions, e.g., the detection of key enzymes associated with the Wood-Ljungdahl pathway. Then, we applied SMMP to investigate vertical variations in the metabolic expression patterns of marine microorganisms from the euphotic zone to the bathypelagic zone. Methane oxidation and carbon monoxide (CO) oxidation were active processes, especially in the bathypelagic zone, which provided a remarkable energy supply for the growth and proliferation of heterotrophic microorganisms. In addition, marker protein profiles detected related to ammonia transport, ammonia oxidation, and carbon fixation highlighted that Thaumarchaeota played a critical role in primary production based on the coupled carbon-nitrogen process, contributing to the storage of carbon and nitrogen in the bathypelagic regions. SMMP has low microbial input requirements and yields in-depth metaproteome analysis, making it a prospective approach for comprehensive marine metaproteomic investigations.
海洋微生物驱动着行星尺度元素循环的关键转化，对全球生物地球化学过程有着至关重要的影响。代谢组学是评估海洋微生物代谢多样性和功能的有力工具。然而，由于海洋微生物种群的稀疏性，通常需要数百升海水进行正常的代谢组学分析，这对海洋代谢组学的广泛应用，特别是对深海海水，构成了巨大的挑战。在此，通过整合聚碳酸酯滤膜辅助微生物富集、基于固相烷基化的抗干扰样品制备以及用于痕量肽分离和鉴定的窄径纳升 LC 柱，开发了一种灵敏的海洋代谢组学工作流程，命名为灵敏海洋代谢组分析（SMMP）。该方法从 1 升深海海水样品中获得了超过 8500 种蛋白质，涵盖了多种微生物和关键功能，例如，检测与伍德-吕荣达尔途径相关的关键酶。然后，我们应用 SMMP 研究了从透光带至深海带的海洋微生物代谢表达模式的垂直变化。甲烷氧化和一氧化碳（CO）氧化是活跃的过程，特别是在深海带，为异养微生物的生长和增殖提供了显著的能量供应。此外，检测到与氨转运、氨氧化和碳固定相关的标记蛋白谱，突出了古菌在基于碳-氮偶联过程的初级生产中发挥着关键作用，有助于碳和氮在深海区的储存。SMMP 对微生物的输入要求低，能进行深入的代谢组学分析，是全面海洋代谢组学研究的一种有前景的方法。
