Carbohydrate epitope analysis (via antibody probing) of POM-derived organic matter in the western North Atlantic Ocean

Collection of particulate organic matter Particulate organic matter (POM) was collected by filtering between 5-15 liters of water through a 47-mm pre-combusted (400℃ for 6 hours) glass fiber filter (GF/F; nominal pore size 0.7 μm; for volumes filtered at each depth and station, see the dataset). Polysaccharide extraction for microarray analyses of POM POM samples were prepared for polysaccharide analysis according to Vidal-Melgosa et al. (2021; ). Polysaccharides were sequentially extracted from four filter piece punches (11.2 mm diameter) from GF/F filters. The samples were first extracted with autoclaved MilliQ water, followed by 50 mM EDTA, and finally 4 M NaOH with 0.1% NaBH4. The supernatant containing extracted polysaccharides was collected from each of the sequential steps and stored at 4 °C. Carbohydrate microarray analysis to determine structural complexity of POM The polysaccharides extracted as described above were analyzed following Vidal-Melgosa et al. (2021). In brief, the polysaccharide extracts were first diluted in printing buffer (55.2% glycerol, 44% water, 0.8% Triton X-100), and then printed on 0.45 µm pore size nitrocellulose membrane (Whatman) using a microarray robot (Sprint, Arrayjet, Roslin, UK) at 20 °C and 50% humidity. The membranes were probed with one of 9 monoclonal antibodies, washed multiple times, and probed with secondary antibodies (anti-rat, anti-mouse, or anti-His tag) conjugated to alkaline phosphatase for 2 hours. The arrays were developed using 5-bromo-4-chloro-3-indolyphosphate and nitro blue tetrazolium in alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl2, 100 mM Tris-HCl, pH 9.5). The microarrays were scanned and signal intensity was acquired using the software Array-Pro Analyzer 6.3 (Media Cybernetics). Signals were normalized among samples; higher signals correspond to a higher abundance of a given polysaccharide epitope. Note that the carbohydrate microarray data are only semiquantitative; while comparisons can be made for the abundance of a given epitope between stations and depths, the signal intensity cannot be used to compare signals of different epitopes.