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.

溶解有机质（Dissolved organic matter，DOM）是一类超复杂混合物，在全球生物地球化学循环中发挥核心作用。尽管其重要性不言而喻，但当前学界对其分子层面的认知仍较为匮乏。近数十年来，学界已通过高分辨质谱（high-resolution mass spectrometry，HR-MS）、液相色谱（liquid chromatography，LC）与串联质谱（tandem mass spectrometry，MS/MS）联用的技术手段，对DOM的化学组成展开了大量解析工作。然而，DOM的复杂性以及大量未被分辨的同分异构体，仍阻碍了其完整结构解析工作的推进。为应对这一挑战，本研究开发了一种离线二维液相色谱（two-dimensional LC，2D-LC）方法：采用两个具备正交pH条件的反相色谱维度，随后进行串联质谱数据采集与分子网络分析。2D-LC-MS/MS技术可降低DOM的复杂度，提升串联质谱谱图质量，并提高谱图注释率。本研究将该方法应用于分析美国南加州海岸表层溶解有机质以及北太平洋中部（夏威夷海域）的开阔大洋溶解有机质，通过串联质谱谱图匹配共注释得到超过600种分子结构，其注释数量较总运行时长相同的迭代一维液相色谱-串联质谱分析提升了最高达90%。本研究的数据为海洋溶解有机质的分子组成分析提供了前所未有的视角，并证实了2D-LC-MS/MS方法在解析超复杂样品化学组成方面的应用潜力。