Metaproteomics analyses of mixed-layer water from Hawaii Ocean Time-series Station ALOHA, collected on R/V Kilo Moana cruise KM2204 in March 2022, and incubated with 15N2 to track biosynthetic incorporation by diazotrophic microbes

This project seeks to shed new light on outstanding questions in marine diazotroph ecology, including: 1) How is whole-community N2 fixation activity apportioned among different diazotroph taxa?; 2) How do diazotrophs and their symbiotic partners make biosynthetic use of the N they fix?; 3) How much diazotroph-derived N is redistributed to particular nondiazotroph taxa? To address these questions, we conducted shipboard field work at Hawaii Ocean Time-series Station ALOHA in the North Pacific, where we sampled mixed-layer ocean surface water microbial communities and performed 15N2-incubation experiments. We developed novel experimental approaches to highly enriching seawater with dissolved 15N2, which is essential for sensitive 15N-tracking proteomics. The protocols we developed were able to achieve >30 atom% 15N in dissolved N2 in shipboard incubations of natural seawater. We used metaproteomics analysis of the microbial biomass from these incubations to assess the N2-fixation activity of coexisting diazotrophic organisms, and to determine the biosynthetic allocation of the fixed nitrogen they produced. These data are available through MassIVE under accession number MSV000096410 (DOI: 10.25345/C5C24R05N).