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

Dissolved organic matter (DOM) is one of the most complex chemical mixtures and plays a central role in biogeochemical cycles across our ecosphere. 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 (HRMS) and liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Yet, the complexity and high degree of non-resolved isomers still hamper the full structural analysis of DOM. To overcome this challenge, we adapted a two-dimensional (2D) LC approach consisting of two reversed-phase dimensions with orthogonal pH, followed by MS/MS data acquisition and molecular networking. The 2D chromatography approach mitigates the complexity of DOM, enhancing both the quality of MS/MS spectra and spectral annotation rates. Applying our approach to analyze coastal surface DOM from Southern California (USA), we annotated in total more than 600 structures via MS/MS spectrum matching, which was up to 90% more than in iterative 1D LC-MS/MS analysis with the same total run time. Our data provide an unprecedented view into the molecular composition of coastal DOM, highlighting the potential of 2D LC-MS/MS approaches to decipher ultra-complex mixtures.