HormonomicsDB: a novel workflow for the untargeted analysis of plant growth regulators and hormones.

Background: Metabolomics is the simultaneous determination of all metabolites in a system. Despite significant advances in the field, compound identification remains a challenge. Prior knowledge of the compound classes of interest can improve metabolite identification. Hormones are a small signaling molecules, which function in coordination to direct all aspects of development, function and reproduction in living systems and which also pose challenges as environmental contaminants. Hormones are inherently present at low levels in tissues, stored in many forms and mobilized rapidly in response to a stimulus making them difficult to measure, identify and quantify. Methods: An in-depth literature review was performed for known hormones, their precursors, metabolites and conjugates in plants to generate the database and an RShiny App developed to enable web-based searches against the database. An accompanying liquid chromatography - mass spectrometry (LC-MS) protocol was developed with retention time prediction in Retip. A meta-analysis of 14 plant metabolomics studies was used for validation. Results: We developed HormonomicsDB, a tool which can be used to query an untargeted mass spectrometry (MS) dataset against a database of more than 200 known hormones, their precursors and metabolites. The protocol encompasses sample preparation, analysis, data processing and hormone annotation and is designed to minimize degradation of labile hormones. The plant system is used a model to illustrate the workflow and data acquisition and interpretation. Analytical conditions were standardized to a 30 min analysis time using a common solvent system to allow for easy transfer by a researcher with basic knowledge of MS. Incorporation of synthetic biotransformations enables prediction of novel metabolites. Conclusions: HormonomicsDB is suitable for use on any LC-MS based system with compatible column and buffer system, enables the characterization of the known hormonome across a diversity of samples, and hypothesis generation to reveal knew insights into hormone signaling networks.