A novel method for efficient screening and annotation of important pathway-associated metabolites based on the modified metabolome and probe molecules

Plants produce a wide variety of secondary metabolites in the process of evolution. Secondary metabolites have highly diverse structures due to the modification of the basic skeletons of metabolites. They are required for interaction with the environment and are produced in response to abiotic/biotic stress. Characterization of secondary metabolic pathways is significant to plant molecular breeding and natural product biosynthesis. The liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) is one of the major techniques for untargeted metabolomics study. The LC-HRMS/MS method could detect tens of thousands of metabolic features and provide abundant structural information. It has been widely used in the discovery and characterization of the secondary metabolome. However, due to the largely diverse structure and limited records in the mass spectral library, the annotation of the secondary metabolome is very difficult. To address the analytical challenges associated with the vast structural diversity and the large numbers of secondary metabolites, particularly those previously unknown structural metabolites, a novel method for the efficient characterization of pathway-associated metabolites was developed. Modification reactions and MS/MS spectral information were collected using the metabolic pathways database and mass spectral library. Screening and annotation of metabolites involved in phenylpropanoid metabolism in maize leaves were used as an example. First, a database of modified groups was established via pathway-associated modifications from open access metabolic pathway database and literature. Here, pathway databases included the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Plant Metabolic Pathways (PlantCyc). A total of 61 modification types were enrolled, including 10 generic and 51 pathway-specific modifications. Modified metabolomes were filtered from untargeted LC-HRMS/MS metabolomics data. Next, MS/MS spectra of the pathway-associated compounds (probe molecules) were collected in the Global Natural Products Social Molecular Networking (GNPS) MS/MS spectral library. The MS/MS of compounds assigned to chemical classes of phenylpropanoids were kept. An MS/MS spectral database of the probe molecules was constructed. It included 2677 spectra of 1542 phenylpropanoid compounds in the positive mode and 814 spectra of 661 phenylpropanoid compounds in the negative mode. Then, an MS/MS molecular network was generated by modified metabolome and probe molecules. The clusters comprising both probe molecules and modified metabolites were kept. To explore more previously unknown structural metabolites, the clusters with one more pathway-specific modified metabolite were retained even though they didn't contain any probe molecule. A total of 392 and 417 phenylpropanoid pathway-related metabolic metabolites were obtained in positive and negative ion modes, respectively. The pathway-associated metabolites were annotated based on the propagation of the molecular network. For the metabolites within the co-cluster, annotations were performed using the probe molecules as the initial seed. The modification group's substructure information was used for network propagation annotation. For the clusters containing only pathway-specific modified metabolites, the annotation is similar to the above process if identified nodes were present within the cluster. Otherwise, de novo annotation was manually executed based on substructure information. Finally, 129 unique metabolites were annotated after integration and removal of redundancy. Ten annotated metabolites were validated using commercially available or synthesized reference compounds. The other annotation results were validated using predicted chemical classes, in silico MS/MS, and predicted retention time. They are mainly involved in the downstream branch of phenylpropanoid pathways, including the flavonoid pathway (8 flavonoids, 19 flavonoid O-glycosides, 32 flavonoid C-glycosides), the hydroxycinnamic acid pathway (31 hydroxycinnamic acids and derivatives), and the lignan pathway (22 neo-lignans/lignan/lignan glycosides). All the annotated structures were searched against the PubChem and SciFinder databases. Among them, 26 metabolites were previously unreported in both the databases. In this study, the pathway-associated metabolites could be quickly discovered and annotated by the integration of probe molecules and modified metabolome. It provides a method for the in-depth study of the phenylpropanoid pathway.