SMMP: A Deep-Coverage Marine Metaproteome Method for Microbial Community Analysis throughout the Water Column Using 1 L of Seawater.

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.