Dataset: 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

Mixed-layer water (25 meters (m) depth) was sampled at Station ALOHA using 12-liter (L) Niskin bottles on the HOT CTD rosette. To prepare 15N₂-enriched seawater for incubations, water was filtered first through nitex mesh (250-micrometers (µm)) and then through 0.2 µm-pore filters (Millipak) into pre-cleaned 2L pyrex glass bottles with gas-port caps (Chemglass). Water was then degassed under vacuum with vigorous stirring (Velp MST magnetic stir plates) for at least 30 minutes. The evacuated headspace was then filled with a slight overpressure of 15N₂ gas (>98 atom% 15N; Cambridge Isotope), which was then allowed to equilibrate with continued vigorous stirring for at least 60 minutes. Incubations were initiated by mixing 2L of mesh-prefiltered mixed-later seawater (sampled from a hydrocast subsequent to that used for the 15N₂-enriched water) in a 1:1 ratio with the 0.2µm-prefiltered 15N₂-enriched water in 4L polycarbonate bottles. Parallel 14N₂-incubations were prepared using 0.2µm-prefiltered, degassed seawater equilibrated with N₂ gas of natural isotopic abundance (0.37 atom% 15N). Bottles were then incubated in on-deck incubators that were temperature controlled with surface seawater from the ship's flow-through system and shaded to mimic the light field at 25m depth.

Samples for proteomic analysis were collected by filtering the contents of the 4L incubation bottles onto 0.2µm-pore PES membrane filters (MilliporeExpress) under gentle suction. Filters were frozen at -80 degrees Celsius (ºC) shipboard immediately upon collection and transported to UChicago in a LN₂-cooled dry shipper for analysis. Proteins were extracted from filters by agitation in a Beadbeater (40 seconds), sonication (QSonica Q500, 10 minutes in 10-second on/off pulses, 85% amplitude), and heating (95ºC, 25 minutes), all in a reducing and denaturing buffer (1% LDS, 200 millimolar (mM) Tris pH 8.0, 10 mM DTT) followed by alkylation of cysteine thiols with 40 mM iodoacetamide. Proteins were then precipitated in 4 volumes of acetone in glass centrifuge tubes overnight at -20ºC and pelleted by centrifugation for 60 minutes at 7000 x g. Protein pellets were dried, redissolved in denaturing buffer (8M urea, 0.2% deoxycholate, 1M ammonium bicarbonate pH 8), and purified using a modified eFASP (enhanced Filter-Aided Sample Preparation) protocol (Erde, et al. 2014) in passivated Sartorius Vivacon 500 concentrators (30 kDa nominal cutoff). Purified proteins were digested on-filter with MS-grade trypsin (37ºC, overnight, 2 micrograms (µg)). Peptides were eluted from the concentrators and dried by vacuum centrifugation.

For LC-MS analysis, peptide samples were reconstituted in 2% acetonitrile + 0.1% formic acid. All samples were separated on a monolithic capillary C18 column (GL Sciences Monocap Ultra, 100µm ID x 200-centimeter (cm) length) using a water-acetonitrile + 0.1% formic acid gradient (2-50% AcN over 180 minutes) at 360 nanoliters per minute (nL/min) using a Dionex Ultimate 3000 LC system with nanoelectrospray ionization (Proxeon Nanospray Flex source). Mass spectra were collected on an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) operating in a data-dependent acquisition mode, with one high-resolution (120,000 m/∆m) MS1 parent ion full scan triggering 15 Rapid mode MS2 CID fragment ion scans of selected precursors.