Recent Developments Toward Integrated Metabolomics Technologies (UHPLC-MS-SPE-NMR and MicroED) for Higher-Throughput Confident Metabolite Identifications

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.

NMR and Metabolomics—A Roadmap for the Future

“…Accordingly, these approaches are directly applicable to metabolomics studies, especially regarding the identification of unknown metabolites. The development of automated SPE systems coupled with MS and NMR has enabled the automated purification of targeted analytes coupled with structure elucidation and/or validation by both MS and NMR [ 121 ]. The use of computer controlled SPE purification and the advanced sensitivity of high-field NMR with cryo-probes or cryo-microprobes have enabled the characterization of microgram quantities of materials, as noted above.…”

“…The use of computer controlled SPE purification and the advanced sensitivity of high-field NMR with cryo-probes or cryo-microprobes have enabled the characterization of microgram quantities of materials, as noted above. Specific examples have included HPLC-UV-SPE-NMR [ 122 , 123 ], HPLC-MS/MS-SPE-NMR [ 106 , 124 ], and UHPLC-MS/MS-SPE-NMR [ 121 , 125 ]. These hyphenated technology ensembles reduce the traditionally lengthy and laborious metabolite discovery and identification process [ 126 ].…”

Electron Diffraction of 3D Molecular Crystals

“…In a field continually grappling with the myriad pitfalls associated with analysis of complex 2D NMR spectra, the clarity provided by a corroborating crystal structure seems almost cathartic. Furthermore, when applied in tandem with comparative genomics or metabolomics (to mine relevant biosynthetic gene clusters) and synthetic biology (to express those genes in model organisms), 3D ED could also significantly accelerate the rate of NP discovery. − More broadly, 3D ED is rapidly finding a complementary niche within the wider context of synthetic chemistry; a growing number of reports now feature 3D ED structures of relevant synthetic targets or intermediates which proved unsuitable for single-crystal XRD. − These structures include two noncanonical amino acids bearing all-carbon quaternary stereocenters, a trio of organic semiconductors solved at ultrahigh resolution (one of which is depicted in Figure E), a family of electron-deficient expanded helicenes, a pentacyclic indole-derived ester, and a synthetic mimic of the cuboidal subunit in the oxygen-evolving complex of photosystem II …”

NMR-Based Chromatography Readouts: Indispensable Tools to “Translate” Analytical Features into Molecular Structures

“…A multitude of publications describe its use to dereplicate the plant extracts of different origin, e.g., from Corydalis solida tubers [ 110 ], from plants with traditional medicinal use such as Harpagophytum procumbens (Devils Claw) [ 86 ], Hypericum perforatum (St. John’s Wort) [ 111 ], Morus alba [ 112 ], or from plants with uncharted bioactivity profiles such as Carthamus oxyacantha [ 113 ] and startling novel fungal compound classes as chiral dispiro-derivatives [ 114 ]. Unraveling the secondary metabolite profile in the model plant Medicago truncatula was facilitated with the aid of this hyphenation [ 115 ] as it was previously described for tomato metabolite profiling [ 116 ]. HPLC-SPE-NMR orientated research is always oriented towards the structural characterization of analytes.…”