Two-Dimensional Liquid Chromatography Tandem Mass Spectrometry Untangles the Deep Metabolome of Marine Dissolved Organic Matter

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