Dataset: Monosaccharide Composition of HMWDOM concentrate and POM-derived Carbohydrates from bulk seawater and mesocosm experiments taken aboard the R/V Endeavor in the Western North Atlantic during the research cruise EN683 in May and June, 2022

Sampling

Glycans were profiled in bulk and high molecular weight dissolved organic matter (HMWDOM) and particulate organic matter (POM) in the deep chlorophyll max (DCM) and bottom water at three stations in the western North Atlantic Ocean. 

Sampling and processing of HMWDOM and POM

Seawater was collected using 30 L Niskin bottles on-board the R/V Endeavor. Niskin bottles were attached to rosettes equipped with conductivity, temperature and depth (CTD) sensors. Physical parameters were recorded using a Seabird 911+ CTD profiler. Data were processed and binned with SBE Data Processing software (v7.26.7). Collected seawater was transferred into acid-washed carboys before processing on-board. HMWDOM was concentrated to ~300 mL by tangential flow filtration (TFF). The TFF system (Sartorius <>) was run with 3 filter cassettes (1 kDa). HMWDOM samples were further concentrated back in the lab by freeze-drying and resuspension in lower volumes of MilliQ-water; for example, 10 mL of HMWDOM was freeze-dried and resuspended in 2.5 to 3mL. These concentrated HMWDOM samples were used for acid hydrolysis and microarray printing. POM samples were taken using in situ pumps onboard R/V Endeavor (EN683). At station 21 ~393 L were filtered in surface waters and 1276 L between 2800 and 3100 m depth. At station 22 ~214 L were sampled at DCM and 931 L at depths between 3321 and 3694 m. At station 23 the in situ pumps were deployed at 110 m (DCM) and 5100 m (depth) and pumped 515 L and 1196 L, respectively. 

Sequential extraction of polysaccharides from GF/F filters 

Seven punchouts of 11 m diameter were made from GF/F filters and sequentially extracted using MilliQ-water, 0.3M EDTA and 4M NaOH + 0.1% NaBH4 (Vidal-Melgosa et al., 2021). Subsequently solvents were added to the filter pieces, vortexed and incubated for 2 h at 60°C at 650 rpm in a heat block. Extracts were centrifuged at 6,000 x g for 15 min at 15°C and the supernatant was transferred to a new tube. NaOH extracts were neutralized with 4M HCl. 

Quantification of monosaccharides 

Polysaccharides of HMWDOM and POM samples were hydrolysed into monosaccharides. Freeze dried and concentrated HMWDOM samples (500 µL) were acid hydrolysed with 500 µL of 2M HCl. Additionally, 10 x 5 mm diameter punchouts from all GF/F filters were directly transferred to ampoules and acid hydrolysed in 750 µL 1M HCl. All hydrolyses were run at 100°C for 24 hrs. The supernatant of acid hydrolysed samples was dried in an acid-resistant vacuum concentrator (Martin Christ Gefriertrocknungsanlagen GmbH, Germany). Hydrolysates were reconstituted in MilliQ-water and pH adjusted to >7 with 0.1 M NaOH. Monosaccharide standards and hydrolysates were spiked with 13C-labelled glucose, galactose and mannose before derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) (Rühmann et al., 2014). PMP-derivatives were separated on a Agilent 1290 Infinity II LC system equipped with a Waters CORTECS UPLC C18 column and measured on a SCIEX qTRAP5500 by multiple reaction monitoring (MRM) (Xu et al., 2018). Signal intensities were normalised to 13-labelled standards and calibrated against standard curves.