Biological Filtering and Substrate Promiscuity Prediction for Annotating Untargeted Metabolomics.

Mass spectrometry coupled with chromatography separation techniques provides a powerful platform for untargeted metabolomics. Determining the chemical identities of detected compounds however remains a major challenge. Here, we present a novel computational workflow, termed extended metabolic model filtering (EMMF), that aims to engineer a candidate set, a listing of putative chemical identities to be used during annotation, through an extended metabolic model (EMM). An EMM includes not only canonical substrates and products of enzymes already cataloged in a database through a reference metabolic model, but also metabolites that can form due to substrate promiscuity. EMMF aims to strike a balance between discovering previously uncharacterized metabolites and the computational burden of annotation. EMMF was applied to untargeted LC-MS data collected from cultures of Chinese hamster ovary (CHO) cells and murine cecal microbiota. EMM metabolites matched, on average, to 23.92% of measured masses, providing a > 7-fold increase in the candidate set size when compared to a reference metabolic model. Many metabolites suggested by EMMF are not catalogued in PubChem. For the CHO cell, we experimentally confirmed the presence of 4-hydroxyphenyllactate, a metabolite predicted by EMMF that has not been previously documented as part of the CHO cell metabolic model.