Click Chemistry-Enabled Screening of Lignin-Derived Compounds via Non-Targeted Metabolomics

Abstract

Lignin-derived compounds (LDCs) are major contributors to soil organic carbon and play a critical role in the global biogeochemical cycle of carbon. However, the reliable identification of LDCs in complex environmental media remains a major analytical challenge. In addition, owing to lignin’s heterogeneous, cross-linked polymeric structure, many LDCs are poorly soluble and exhibit low ionization efficiency in mass spectrometry, substantially hindering their identification. To overcome these analytical hurdles, we developed an advanced analytical pipeline integrating click reaction with high-performance liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMS/MS) and nontargeted metabolomic analysis, enabling the systematic screening and putative identification of LDCs, including species that are otherwise poorly ionizable or insoluble. Our study screened LDCs by reacting extractions of azide-tagged lignin polymers with dibenzocyclooctyne-polyethylene glycol acid (DBCO-PEG1). We employed two complementary analytical pathways: (i) targeted mass difference screening of azide-tagging and DBCO-PEG1 adduct, and (ii) diagnostic MS/MS fingerprint analysis based on characteristic fragment ions of DBCO-PEG1. Using this strategy, we screened 17 high-confidence triplet candidates of LDCs and additional putative candidates identified via MS/MS fingerprinting, spanning a broad chemical space. By integrating click reaction with HPLC-HRMS/MS and nontargeted metabolomic analysis, this study established a framework for tagging and screening analytically inaccessible (“dark”) organic compounds. Further structural characterization of candidate compounds and the application of this approach to incubation studies provides a pathway toward improved molecular identification of LDCs crucial to soil organic carbon persistence.