Two-Dimensional\nLiquid Chromatography Tandem Mass\nSpectrometry Untangles the Deep Metabolome of Marine Dissolved Organic\nMatter

Dissolved organic matter (DOM) is an ultracomplex mixture\nthat\nplays a central role in global biogeochemical cycles. Despite its\nimportance, DOM remains poorly understood at the molecular level.\nOver the last decades, significant efforts have been made to decipher\nthe chemical composition of DOM by high-resolution mass spectrometry\n(HR-MS) and liquid chromatography (LC) coupled with tandem mass spectrometry\n(MS/MS). Yet, the complexity and high degree of nonresolved isomers\nstill hamper the full structural analysis of DOM. To address this\nchallenge, we developed an offline two-dimensional (2D) LC approach\nusing two reversed-phase dimensions with orthogonal pH levels, followed\nby MS/MS data acquisition and molecular networking. 2D-LC-MS/MS reduced\nthe complexity of DOM, enhancing the quality of MS/MS spectra and\nincreasing spectral annotation rates. Applying our approach to analyze\ncoastal-surface DOM from Southern California (USA) and open-ocean\nDOM from the central North Pacific (Hawaii), we annotated in total\nmore than 600 structures via MS/MS spectrum matching, which was up\nto 90% more than that in iterative 1D LC-MS/MS analysis with the same\ntotal run time. Our data offer unprecedented insights into the molecular\ncomposition of marine DOM and highlight the potential of 2D-LC-MS/MS\napproaches to decipher the chemical composition of ultracomplex samples.