Water samples were collected in August of 2013 using a 24 bottle (10 L General Oceanics Niskin X) rosette equipped with CTD sensors; 160 liters of water were collected from the Bering Strait (BSt) chlorophyll maximum layer (7 m depth; 65° 43.44”N, 168° 57.42”W). The integrated initial Chl a measurement was 226.88 mg/m2, temperature was 2.06°C and salinity was 32.15.

Water was prefiltered to remove all eukaryotic grazers and particles by sequential filtration: 10.0 µm followed by a 1.0 µm polycarbonate (PC) filter. For the generation of a site/time-specific metagenome, seven 1-L aliquots of 1.0 µm prefiltered water was filtered onto 0.2 µm PC filters for a total of 7 filters. Metaproteomics samples of free-living ocean microbes were collected on 0.2 µm Nuclepore Track-Etch membrane PC filters (Whatman, Maidstone, UK) from shipboard incubations at 0 ºC in the dark over 10 days (T0 = day 0, T10 = day 10). The 10-day incubation was designed to follow the changes in community composition and metabolism with and without an exogenous source of carbon. Upon collection, all filters were immediately frozen in liquid nitrogen and stored at -80 ºC. In the full experiment, multiple parameters (in addition to proteomics) were measured at several time points over the 10-day incubation (manuscript in prep.). There are, of course, limitations to measuring community function during an incubation in a closed system, but those limitations will be addressed more fully in the biologically- and ecologically-focused future manuscript.

Filters for the metaproteomics analysis were sliced and submerged in 100 µl of 6 M urea and 600 µl of 50 mM NH4HCO3. Whole cell lysing was accomplished with a Branson 250 probe sonicator (duty cycle 40%; output control 3) for 20 seconds (Branson Ultrasonics, Danbury, CT). Between sonication events, each filter was flash-frozen in liquid nitrogen to reduce protease activity. Protease inhibitors are avoided because they interfere with downstream digestion and mass spectrometry. This sonication-freezing cycle was repeated five times. Filters were rinsed two times in 100 µl of ultrapure water to remove remaining cellular debris from the filter. Protein extraction and desalting followed the protocol outlined in Nunn et al., (2015).

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was done on a nanoAcquity UPLC (Waters Corp, Milford, MA) connected to a Q-Exactive-HF (Thermo Fisher Scientific, Waltham, MA) on technical duplicates for each sample. A total of 11 mixed microbial samples were analyzed.

Analytical and trapping columns were made and packed in-house. Laser-pulled analytical columns were packed with 3 µm C18 beads (Dr. Maisch HPLC GmBH, Germany) to 15 cm (75 µm ID) and 2 cm trapping columns (100 µm ID) were packed with 3 µm C12 beads (Dr. Maisch) with a Kasil-frit. Peptides were eluted from the column with 5%-30% ACN, 0.1% formic acid in 90 minutes using a 300 nl/min flow rate. The mass spectrometer was operated in Top 20 DDA mode with an MS1 scan range of 400-1000 m/z, 15 second dynamic exclusion, and to increase sensitivity of microbial peptides, 5 trypsin peptide ions were added to the exclusion list (421.7598, 842.5121, 532.2871, and 1045.5664). Mass accuracy and chromatographic retention time were monitored using a quality control standard of 15 peptides (PRTC-BSA, Thermo Fisher Scientific).

Instrument(s): QExactive Thermo Finnegan: DDA data

BeringSt_Incubations_Mikan_Timmins.csv