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What Is Widely Targeted Metabolomics
Plant untargeted metabolomics commonly employs high-resolution mass spectrometry (qTOF/Orbitrap) for qualitative analysis of metabolites. It offers high throughput and high resolution in secondary spectrum, but is limited by dynamic range and sensitivity. Targeted metabolomics, on the other hand, utilizes standard reference materials for qualitative and quantitative analysis of metabolites. It has a wide dynamic range and high sensitivity, but lower throughput.
Plant widely targeted metabolomics, distinct from existing metabolite detection methods, is a novel metabolomics approach that integrates the advantages of "comprehensiveness" from untargeted metabolomics and "accuracy" from targeted metabolomics. It features high throughput, ultra-sensitivity, broad coverage, and accurate qualitative and quantitative analysis. Specifically designed for plant samples, it enables the simultaneous qualitative and quantitative detection of thousands of metabolites in plant samples by constructing a plant-specific metabolite database. This provides a more efficient method for the detection of complex plant metabolites and serves as a crucial foundation for studying metabolic pathways and biological mechanisms.
Advantages Of Widely Targeted Metabolomics
Comprehensive substance identification: High throughput, simultaneous qualitative and quantitative detection of 1000+ metabolites.
Comprehensive annotation information: Over 90% KEGG coverage, over 90% coverage of primary and secondary metabolite classification, over 90% annotation of compound names. Accurate and reliable pathway analysis and functional analysis. Reliable correlation analysis through multi-omics integration.
Wide coverage of substance categories: Construction of libraries through repeated testing using multiple species and sample types. Multiple preprocessing methods employed for different compound characteristics. High coverage of plant metabolites.
Large number of measured metabolites in projects: For example, a project measured over 700-1300 plant metabolites, with 870+ substances detected (including 250+ flavonoids, 120+ alkaloids, 90+ polyphenols, 110+ terpenes, 300+ coumarins and other categories, 50+ miscellaneous).
High sensitivity: AB Q Trap 6500+ MRM enables detection of low-abundance secondary metabolites that cannot be detected by un-targeted techniques using data scanning modes.
Accurate qualitative analysis: Compared to un-targeted techniques, comprehensive targeted metabolomics utilizes Q1, Q3, CE, and DP for qualitative analysis.
Comprehensive database: Establishment of a plant-specific self-built database with over 10,000 substances, covering primary and secondary metabolites.
Good reproducibility: Generally, 3 biological replicates are sufficient, whereas un-targeted approaches require 6-30 biological replicates.
Plant Widely Targeted Metabolomics Database
| Category | Number | Representative Compounds |
|---|---|---|
| Amino Acids | 300+ | L-Arginine, L-Valine, L-Isoleucine, L-Glutamine, L-Leucine, L-Threonine, D-Glutamic acid, L-Alanine |
| Phenols | 120+ | Methyl caffeate, Eugenol, Lactucin, Fenchyl alcohol, Isoeugenol, Danshenol, Geraniol, Lithospermic acid |
| Phenolic acids | 1200+ | Gastrodin, Ferulic acid, Danshensu, Gingerol, Arbutin, Morroniside, Chlorogenic acid, Glycyrrhetinic acid |
| Nucleotides | 200+ | Adenosine, Cytidine, Guanosine, Uracil nucleoside, 5'-Triphosphate cytidine, 2-Thiocytidine, Cyclic adenosine monophosphate |
| Flavonoids | 2000+ | Mangiferin, Luteolin, Quercetin, Kaempferol, Astragaloside IV, Hesperidin, Cyanidin-3-O-glucoside, Silybin, Malvidin-3-O-glucoside |
| Quinones | 170+ | Deoxyrhapontin, Mitoxantrone, Aloe-emodin, Juglone, 1,4-Naphthoquinone, Emodin, Danthron, Anthraquinone |
| Terpenes | 270+ | Phyllanthin, Camphor, Schisandrin E, Gypenoside, Acteoside, Ligustroside, Magnolol, Honokiol |
| Alkaloids | 1300+ | Taxane, Uncaria alkaloids, Corydaline, Dendrobine, Theobromine, Stepholidine, Matrine, Berbamine |
| Sugars and Polyols | 120+ | L-(-)-Arabinose, β-Cyclodextrin, Trehalose, Naphthalenediol, Maltotetraose, Sorbitol, Sucrose |
| Terpenoids | 1200+ | Huperzine A, Forsythiaside IIA, Cudrania tricuspidata, Campylotropis hirtella saponin, Hericenone, Ginsenoside Rh2, Dihydroartemisinin |
| Ketones, Aldehydes, Acids | 330+ | Vanillin, Matricin, Ethyl vanillin, Lobeline, Methyl heptenone, Triptolide, Succinic acid, Pyridoxal |
| Vitamins | 10+ | Vitamin E, Vitamin K4, Vitamin P4, Pyridoxol, α-Tocopherol, Vitamin B12, Pyridoxal, Retinal |
| Coumarins | 270+ | Piperine, Fraxetin, Ligustilide, Isopropylantipyrine, Precursor D, Trimethylammonium, 5,7-Dihydroxy-4-methylcoumarin, Citrus aurantium |
| Oxygenated anthraquinones | 20+ | Diosgenin, Isohemsleyanin, Isorhapontin, Quercetin-3-glucoside, Iridin, Irisquinone, Demethylsuberosin, Morin |
| Organic acids | 370+ | Dehydroandrographolide, Lacquer tree acid, Picroside II, Aconitic acid, Veratric acid, Ginkgolic acid, Veratramine, Resorcinol |
| Steroids | 200+ | Paeoniflorin, Glycyrrhizin, Hyperin, Gentianose, Atractyloside, Abietic acid, Suillin, Cortisone |
| Lipids | 450+ | Capric acid, Tetradecanedioic acid, Tridecanoic acid, Undecanedioic acid, 10-Undecenoic acid, Ricinoleic acid, Oleic acid, Lauric acid |
| Others | 1300+ | Rubidium G, Xanthocurcumine, Isoborneol, Ethyl citrate, Lucidin, Jasmine ketone, Methyl vanillate, Ether mycin |
| 10000+ |
Workflow Of Widely Targeted Metabolomics
Technical Parameters
Plant tissue: Fresh sample ≥ 2g, dried sample ≥ 1g, freeze-dried powder 250mg
Storage and transportation: Store in liquid nitrogen or at -80°C; Sufficient dry ice for transportation to avoid repeated freeze-thaw cycles Detection platform: Qtrap 6500+, Sciex
Applications Of Widely Targeted Metabolomics In Plants
- Identification of metabolite composition in plant samples
- Study of major secondary metabolic pathways in plants
- Research on plant defense mechanisms against insects and diseases
- Investigation of plant stress resistance mechanisms (salt, drought, pests, etc.)
- Study of plant-microbe interactions
- Identification of components in root exudates
Integrated Metabolomics and Transcriptomics Analyses Revealthe Metabolic Differences and Molecular Basis of NutritionalQuality in Landraces and Cultivated Rice
Journal: Metabolites
Published: 2022
Technical platforms: widely targeted metabolomics, transcriptomics, recombinant protein expression, and in vitro enzyme activity analysis.
Materials: 114 rice germplasm resources, including 35 local varieties and 79 cultivated varieties.
Results
A widely metabolomic approach was utilized to construct a rice seed metabolome database, which comprises 985 high-quality metabolic signals. A total of 549 metabolites were identified through standard compound analysis and database matching. Principal component analysis (PCA), heatmap clustering, and classification analysis revealed 90 significantly different metabolites between cultivated and local rice seeds. Compared to cultivated rice seeds, local varieties exhibited higher levels of anthocyanins and flavonoids compounds.
2 Metabolomic analysis was performed on seeds of four cultivated varieties and six local varieties. Analysis using PCA and Venn diagrams revealed that seed color may be a contributing factor to the metabolic differences during germination between different varieties and localities. Transcriptomic sequencing analysis showed that differentially expressed genes (DEGs) were mainly enriched in phenylpropane metabolism and amino acid metabolism. Integrated analysis of transcriptomics and metabolomics indicated significant differences in the accumulation of flavonoids and anthocyanins during seed germination in local varieties.
3 Investigation of the diversity and molecular mechanisms of anthocyanin metabolism in different types of rice seeds: Construction of expression vectors for relevant proteins and conducting in vitro enzyme activity assays. The levels of anthocyanins and key genes involved in anthocyanin synthesis were found to be higher in local varieties compared to cultivated rice.
Conclusion
This study demonstrated the crucial role of secondary metabolites in the germination process of colored rice seeds through widely targeted metabolomics analysis. By identifying differentially accumulated secondary metabolites, particularly flavonoids and anthocyanins, in local varieties during seed germination, it provides a foundation for further research on the nutritional quality mechanisms of local rice varieties.