High-field NMR spectroscopy and FTICR mass spectrometry: powerful discovery tools for the molecular level characterization of marine dissolved organic matter

High-performance, non-target, high-resolution organic structural spectroscopy was applied to solid phase extracted marine dissolved organic matter (SPE-DOM) isolated from four different depths in the open South Atlantic Ocean off the Angola coast (3° E, 18° S; Angola Basin) and provided molecular level information with extraordinary coverage and resolution. Sampling was performed at depths of 5 m (Angola Current; near-surface photic zone), 48 m (Angola Current; fluorescence maximum), 200 m (still above Antarctic Intermediate Water, AAIW; upper mesopelagic zone) and 5446 m (North Atlantic Deep Water, NADW; abyssopelagic, ~30 m above seafloor) and produced SPE-DOM with near 40% carbon yield and beneficial nuclear magnetic resonance (NMR) relaxation properties, a crucial prerequisite for the acquisition of NMR spectra with excellent resolution. 1H and 13C NMR spectra of all four marine SPE-DOM showed smooth bulk envelopes, reflecting intrinsic averaging from massive signal overlap, with a few percent of visibly resolved signatures and variable abundances for all major chemical environments. The abundance of singly oxygenated aliphatics and acetate derivatives in 1H NMR spectra declined from surface to deep marine SPE-DOM, whereas C-based aliphatics and carboxyl-rich alicyclic molecules (CRAM) increased in abundance. Surface SPE-DOM contained fewer methyl esters than all other samples, likely a consequence of direct exposure to sunlight. Integration of 13C NMR spectra revealed continual increase of carboxylic acids and ketones from surface to depth, reflecting a progressive oxygenation, with concomitant decline of carbohydrate-related substructures. Aliphatic branching increased with depth, whereas the fraction of oxygenated aliphatics declined for methine, methylene and methyl carbon. Lipids in the oldest SPE-DOM at 5446 m showed a larger share of ethyl groups and methylene carbon than observed in the other samples. Two-dimensional NMR spectra showed exceptional resolution and depicted resolved molecular signatures in excess of a certain minimum abundance. Classical methyl groups terminating aliphatic chains represented ~15% of total methyl in all samples investigated. A noticeable fraction of methyl (~2%) was bound to olefinic carbon. Methyl ethers were abundant in surface marine SPE-DOM, and the chemical diversity of carbohydrates was larger than that of freshwater and soil DOM. In all samples, we identified sp2-hybridized carbon chemical environments with discrimination of isolated and conjugated olefins and α,β-unsaturated double bonds. Olefinic proton and carbon atoms were more abundant than aromatic ones; olefinic unsaturation in marine SPE-DOM will be more directly traceable to ultimate biogenic precursors than aromatic unsaturation. The abundance of furan, pyrrol and thiophene derivatives was marginal, whereas benzene derivatives, phenols and six-membered nitrogen heterocycles were prominent; a yet unassigned set of six-membered N-heterocycl