Abstract
Metabolomics has emerged as a powerful discipline to study complex biological systems from a small molecule perspective. The success of metabolomics hinges upon reliable annotations of spectral features obtained from MS and/or NMR. In spite of tremendous progress with regards to analytical instrumentation and computational tools, < 20% of spectral features are confidently identified in most untargeted metabolomics experiments. This article explores the integration of multiple analytical instruments such as UHPLC-MS/MS-SPE-NMR and the cryo-EM method MicroED to achieve large-scale and confident metabolite identifications in a higher-throughput manner. UHPLC-MS/MS-SPE allows for the simultaneous automated purification of metabolites followed by offline structure elucidation and structure validation by NMR and MicroED. Large-scale study of complex metabolomes such as that of the model plant legume Medicago truncatula can be achieved using an integrated UHPLC-MS/MS-SPE-NMR metabolomics platform. Additionally, recent developments in MicroED to study structures of small organic molecules have enabled faster, easier and precise structure determinations of metabolites. A MicroED small molecule structure elucidation workflow (e.g., crystal screening, sample preparation, data collection and data processing/structure determination) has been described. Ongoing MicroED methods development and its future scope related to structure elucidation of specialized metabolites and metabolomics are highlighted. The incorporation of MicroED with a UHPLC-MS/MS-SPE-NMR instrumental ensemble offers the potential to accelerate and achieve higher rates of metabolite identification.
Highlights
Metabolomics is the large-scale profiling of metabolites and it has been applied to a multitude of biological systems
All authors provided editorial feedback, revisions and approved the manuscript. Integrated analytical platforms such as ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry (MS)-solid phase extraction (SPE) coupled with nuclear magnetic resonance (NMR) and microcrystal electron diffraction (MicroED) offer alternative and effective approaches toward identification of metabolites that often remain unidentified in conventional metabolomics database search workflows
Introduction of MicroED into the liquid chromatography (LC)-MS-SPE purification and structure elucidation pipeline is especially exciting as it opens up new avenues for faster, easier and higher-throughput identification of metabolites
Summary
Metabolomics is the large-scale profiling of metabolites and it has been applied to a multitude of biological systems. We believe that the incorporation of MicroED into the UHPLC-MS/ MS-SPE workflow might lead to faster and higher-throughput identifications of biologically important unidentified metabolites Development of these integrated metabolomics platforms can especially benefit the plant and microbial natural products community where large-scale identification of specialized metabolites is a major challenge due to the vast chemical diversity of known as well as truly novel compounds. MicroED is likely to solve the structures from powder mixtures on the same grid, which was demonstrated using mixtures of biotin, carbamazepine, cinchonine, and brucine powders (Jones et al, 2018) These initial studies indicate that MicroED can be a powerful tool in small molecule structure elucidation and can potentially accelerate compound identification in the natural products and metabolomics community.
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