Award: OCE-2050685

Nitrogen fixation, the biological process by which atmospheric nitrogen gas (N2) is converted to ammonium that cells can use to build biomolecules such as proteins, is the largest input of nutrient nitrogen (N) to the ocean. Despite its global importance, there are many unknowns concerning which microorganisms perform much of this nitrogen fixation, how this N is used by the cells that fix it, and how it goes on to feed the nutrient needs of the broader ocean ecosystem. Since most nutrient N is used by microorganisms to build proteins, an ideal way to address these knowledge gaps would be to track nitrogen atoms as they are transformed from dissolved N2 into proteins, whose amino acid sequences can be used to identify which organisms are using the N2-derived nitrogen. In this project, we developed a technique, 15N2-tracking proteomics, to do just that, by adding N2 that is enriched in the naturally rare stable isotope of nitrogen, nitrogen-15, and then measuring the biosynthetic incorporation of that isotope by mass spectrometry-based proteomics. We performed shipboard 15N2 incubation experiments on two research cruises at the Hawaii Ocean Time-series (HOT) oceanographic research station in the north Pacific. On these cruises, we tested two methods pre-enrichment of degassed water and bubble-equilibration with rotational agitation for introducing dissolved 15N2 into natural seawater, and found that both methods enable high (30 atom%) 15N enrichment of dissolved N2. Our 15N2-tracking proteomics data revealed how the active nitrogen-fixing community shifts seasonally between early spring and late summer as the North Pacific water column becomes increasingly stratified and oligotrophic. Overall, this work establishes isotope-tracking proteomics as a valuable addition to our analytical toolkit for understanding the nitrogen cycle in the ocean, particularly for assessment of the taxon-specific N2 fixation activity of marine microorganisms. Last Modified: 05/15/2024 Submitted by: JacobWaldbauer