Summary of Study ST004151

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002613. The data can be accessed directly via it's Project DOI: 10.21228/M8WK0T This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST004151
Study TitleDevelopment of plant extracts as substrates for untargeted transporter substrate identification in Xenopus oocytes
Study SummaryTo expand chemical space of plant extracts for transporter assay, we prepared Arabidopsis seedlings extracts from liquid culture-grown plants subjected to biotic stress (flagellin 22 and chitin treatments to mimic bacterial and fungal infections) and abiotic stress (phosphorus and nitrogen starvation). Extracts from these treatments were characterized and subsequently combined into a single metabolic mixture to capture treatment-specific metabolites and expand the metabolic space. Toxicity testing of the pooled extract against X. laevis oocytes revealed that a liquid-liquid extraction protocol, facilitating lipophilic compound removal, outperformed single solvent extractions in terms of metabolite repeatability and reduced membrane permeation. Metabolite profiling of the final extract using pure standards, structural databases, and in-silico tools identified over 200 metabolites. Our study highlights the importance of developing metabolically diverse yet low-toxicity plant extracts as a critical step toward advancing plant transporter substrate screening. The optimized extraction protocol in combination with X. laevis oocyte assays, provide a robust platform for the functional characterization of plant transporters, paving the way for deeper insights into plant physiology and metabolism.
Institute
University of Copenhagen
DepartmentPlant Molecular Biology
Last NameTheodorou
First NameChristos
AddressThorvaldsensvej 40, Frederiksberg C
Emailctheodot@plen.ku.dk
Phone+4552630800
Submit Date2025-08-22
Raw Data AvailableYes
Raw Data File Type(s)mzML, d(Bruker), raw(Waters)
Analysis Type DetailLC-MS
Release Date2025-09-02
Release Version1
Christos Theodorou Christos Theodorou
https://dx.doi.org/10.21228/M8WK0T
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002613
Project DOI:doi: 10.21228/M8WK0T
Project Title:Development of plant extracts as substrates for untargeted transporter substrate identification in Xenopus oocytes
Project Type:Research article
Project Summary:Amongst the thousands of transport proteins constituting approximately 10% of coding sequences in a genome, only few have an assigned function. Heterologous expression of transporters in combination with the use of plant extracts as complex mixtures of substrates can be a powerful tool for untargeted identification of plant transporter functionality. In this study, we developed and evaluated four extraction protocols to generate Arabidopsis thaliana seedling extracts for use as substrate mixtures in high-throughput screening of transporters expressed in Xenopus laevis oocytes, a well-established system for transporter studies. Our study highlights the importance of developing metabolically diverse yet low-toxicity plant extracts as a critical step toward advancing plant transporter substrate screening. The optimized extraction protocol in combination with X. laevis oocyte assays provide a robust platform for the functional characterization of plant transporters, paving the way for deeper insights into plant physiology and metabolism.
Institute:University of Copenhagen
Department:Plant Molecular Biology
Last Name:Theodorou
First Name:Christos
Address:Thorvaldsensvej 40, Copenhagen, Frederiksberg C, 1871, Denmark
Email:ctheodot@plen.ku.dk
Phone:52630800
Funding Source:The present work was supported by The Villum Foundation (#37798) grant awarded to Villum Investigator Professor Barbara Ann Halkier.

Subject:

Subject ID:SU004302
Subject Type:Plant
Subject Species:Arabidopsis thaliana
Gender:Not applicable
Species Group:Treated and untreated plants

Factors:

Subject type: Plant; Subject species: Arabidopsis thaliana (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment Day Extraction Protocol
SA48040118_FDDAnArabidopsis Thaliana Chitin Non relevant D
SA48040218_FDDApArabidopsis Thaliana Chitin Non relevant D
SA48040317_FDDApArabidopsis Thaliana Chitin Non relevant D
SA48040419_FDDApArabidopsis Thaliana Chitin Non relevant D
SA48040517_FDDAnArabidopsis Thaliana Chitin Non relevant D
SA48040620_FDDApArabidopsis Thaliana Chitin Non relevant D
SA48040719_FDDAnArabidopsis Thaliana Chitin Non relevant D
SA48040820_FDDAnArabidopsis Thaliana Chitin Non relevant D
SA48040916_FDDApArabidopsis Thaliana Flagellin 22 Non relevant D
SA48041015_FDDApArabidopsis Thaliana Flagellin 22 Non relevant D
SA48041114_FDDApArabidopsis Thaliana Flagellin 22 Non relevant D
SA48041213_FDDApArabidopsis Thaliana Flagellin 22 Non relevant D
SA48041313_FDDAnArabidopsis Thaliana Flagellin 22 Non relevant D
SA48041415_FDDAnArabidopsis Thaliana Flagellin 22 Non relevant D
SA48041514_FDDAnArabidopsis Thaliana Flagellin 22 Non relevant D
SA48041616_FDDAnArabidopsis Thaliana Flagellin 22 Non relevant D
SA48041712_FDDAnArabidopsis Thaliana N-starvation Non relevant D
SA48041811_FDDAnArabidopsis Thaliana N-starvation Non relevant D
SA48041909_FDDApArabidopsis Thaliana N-starvation Non relevant D
SA48042011_FDDApArabidopsis Thaliana N-starvation Non relevant D
SA48042112_FDDApArabidopsis Thaliana N-starvation Non relevant D
SA48042210_FDDApArabidopsis Thaliana N-starvation Non relevant D
SA48042310_FDDAnArabidopsis Thaliana N-starvation Non relevant D
SA480432QC1_FDDApArabidopsis Thaliana Pooled Non relevant D
SA480433QC2_FDDApArabidopsis Thaliana Pooled Non relevant D
SA480434QC3_FDDApArabidopsis Thaliana Pooled Non relevant D
SA480435QC4_FDDApArabidopsis Thaliana Pooled Non relevant D
SA480436QC1_FDDAnArabidopsis Thaliana Pooled Non relevant D
SA480437QC2_FDDAnArabidopsis Thaliana Pooled Non relevant D
SA480438QC3_FDDAnArabidopsis Thaliana Pooled Non relevant D
SA480439QC4_FDDAnArabidopsis Thaliana Pooled Non relevant D
SA48042408_FDDApArabidopsis Thaliana P-starvation Non relevant D
SA48042507_FDDApArabidopsis Thaliana P-starvation Non relevant D
SA48042605_FDDAnArabidopsis Thaliana P-starvation Non relevant D
SA48042705_FDDApArabidopsis Thaliana P-starvation Non relevant D
SA48042806_FDDAnArabidopsis Thaliana P-starvation Non relevant D
SA48042907_FDDAnArabidopsis Thaliana P-starvation Non relevant D
SA48043008_FDDAnArabidopsis Thaliana P-starvation Non relevant D
SA48043106_FDDApArabidopsis Thaliana P-starvation Non relevant D
SA48044001-CT240125-EA-REP1 pos_RB1_01_44073_modifiedArabidopsis Thaliana Untreated 1 A
SA48044101-CT240125-EA-REP1 neg_RB1_01_44110_modifiedArabidopsis Thaliana Untreated 1 A
SA48044205-CT240125-EA-REP5 neg_RB5_01_44107_modifiedArabidopsis Thaliana Untreated 1 A
SA48044303-CT240125-EA-REP3 neg_RB3_01_44118_modifiedArabidopsis Thaliana Untreated 1 A
SA48044405-CT240125-EA-REP5 pos_RB5_01_44057_modifiedArabidopsis Thaliana Untreated 1 A
SA48044504-CT240125-EA-REP4 neg_RB4_01_44122_modifiedArabidopsis Thaliana Untreated 1 A
SA48044603-CT240125-EA-REP3 pos_RB3_01_44071_modifiedArabidopsis Thaliana Untreated 1 A
SA48044702-CT240125-EA-REP2 neg_RB2_01_44109_modifiedArabidopsis Thaliana Untreated 1 A
SA48044804-CT240125-EA-REP4 pos_RB4_01_44063_modifiedArabidopsis Thaliana Untreated 1 A
SA48044902-CT240125-EA-REP2 pos_RB2_01_44072_modifiedArabidopsis Thaliana Untreated 1 A
SA48045006-CT240125-EB-REP1 pos_RB6_01_44059_modifiedArabidopsis Thaliana Untreated 1 B
SA48045108-CT240125-EB-REP3 pos_RB8_01_44074_modifiedArabidopsis Thaliana Untreated 1 B
SA48045207-CT240125-EB-REP2 pos_RB7_01_44054_modifiedArabidopsis Thaliana Untreated 1 B
SA48045309-CT240125-EB-REP4 pos_RC1_01_44067_modifiedArabidopsis Thaliana Untreated 1 B
SA48045406-CT240125-EB-REP1 neg_RB6_01_44124_modifiedArabidopsis Thaliana Untreated 1 B
SA48045507-CT240125-EB-REP2 neg_RB7_01_44111_modifiedArabidopsis Thaliana Untreated 1 B
SA48045609-CT240125-EB-REP4 neg_RC1_01_44123_modifiedArabidopsis Thaliana Untreated 1 B
SA48045710-CT240125-EB-REP5 pos_RC2_01_44055_modifiedArabidopsis Thaliana Untreated 1 B
SA48045808-CT240125-EB-REP3 neg_RB8_01_44120_modifiedArabidopsis Thaliana Untreated 1 B
SA48045910-CT240125-EB-REP5 neg_RC2_01_44114_modifiedArabidopsis Thaliana Untreated 1 B
SA48046012-CT240125-EC-REP2 neg_RC4_01_44112_modifiedArabidopsis Thaliana Untreated 1 C
SA48046115-CT240125-EC-REP5 neg_RC7_01_44106_modifiedArabidopsis Thaliana Untreated 1 C
SA48046211-CT240125-EC-REP1 neg_RC3_01_44127_modifiedArabidopsis Thaliana Untreated 1 C
SA48046315-CT240125-EC-REP5 pos_RC7_01_44070_modifiedArabidopsis Thaliana Untreated 1 C
SA48046413-CT240125-EC-REP3 neg_RC5_01_44125_modifiedArabidopsis Thaliana Untreated 1 C
SA48046514-CT240125-EC-REP4 pos_RC6_01_44056_modifiedArabidopsis Thaliana Untreated 1 C
SA48046614-CT240125-EC-REP4 neg_RC6_01_44119_modifiedArabidopsis Thaliana Untreated 1 C
SA48046713-CT240125-EC-REP3 pos_RC5_01_44060_modifiedArabidopsis Thaliana Untreated 1 C
SA48046811-CT240125-EC-REP1 pos_RC3_01_44053_modifiedArabidopsis Thaliana Untreated 1 C
SA48046912-CT240125-EC-REP2 pos_RC4_01_44062_modifiedArabidopsis Thaliana Untreated 1 C
SA48047020-CT240125-ED-REP5 neg_RD4_01_44126_modifiedArabidopsis Thaliana Untreated 1 D
SA48047116-CT240125-ED-REP1 pos_RC8_01_44068_modifiedArabidopsis Thaliana Untreated 1 D
SA48047219-CT240125-ED-REP4 neg_RD3_01_44108_modifiedArabidopsis Thaliana Untreated 1 D
SA48047317-CT240125-ED-REP2 pos_RD1_01_44065_modifiedArabidopsis Thaliana Untreated 1 D
SA48047418-CT240125-ED-REP3 neg_RD2_01_44116_modifiedArabidopsis Thaliana Untreated 1 D
SA48047520-CT240125-ED-REP5 pos_RD4_01_44064_modifiedArabidopsis Thaliana Untreated 1 D
SA48047618-CT240125-ED-REP3 pos_RD2_01_44066_modifiedArabidopsis Thaliana Untreated 1 D
SA48047717-CT240125-ED-REP2 neg_RD1_01_44117_modifiedArabidopsis Thaliana Untreated 1 D
SA48047819-CT240125-ED-REP4 pos_RD3_01_44058_modifiedArabidopsis Thaliana Untreated 1 D
SA48047916-CT240125-ED-REP1 neg_RC8_01_44115_modifiedArabidopsis Thaliana Untreated 1 D
SA48048002-CT240125-EA-REP2 neg_RB2_01_44519_modifiedArabidopsis Thaliana Untreated 2 A
SA48048104-CT240125-EA-REP4 neg_RB4_01_44532_modifiedArabidopsis Thaliana Untreated 2 A
SA48048205-CT240125-EA-REP5 neg_RB5_01_44517_modifiedArabidopsis Thaliana Untreated 2 A
SA48048303-CT240125-EA-REP3 neg_RB3_01_44528_modifiedArabidopsis Thaliana Untreated 2 A
SA48048401-CT240125-EA-REP1 neg_RB1_01_44520_modifiedArabidopsis Thaliana Untreated 2 A
SA48048505-CT240125-EA-REP5 pos_RB5_01_44467_modifiedArabidopsis Thaliana Untreated 2 A
SA48048604-CT240125-EA-REP4 pos_RB4_01_44473_modifiedArabidopsis Thaliana Untreated 2 A
SA48048701-CT240125-EA-REP1 pos_RB1_01_44483_modifiedArabidopsis Thaliana Untreated 2 A
SA48048803-CT240125-EA-REP3 pos_RB3_01_44481_modifiedArabidopsis Thaliana Untreated 2 A
SA48048902-CT240125-EA-REP2 pos_RB2_01_44482_modifiedArabidopsis Thaliana Untreated 2 A
SA48049010-CT240125-EB-REP5 neg_RC2_01_44524_modifiedArabidopsis Thaliana Untreated 2 B
SA48049109-CT240125-EB-REP4 neg_RC1_01_44533_modifiedArabidopsis Thaliana Untreated 2 B
SA48049208-CT240125-EB-REP3 neg_RB8_01_44530_modifiedArabidopsis Thaliana Untreated 2 B
SA48049307-CT240125-EB-REP2re pos_RB7_01_44464_modifiedArabidopsis Thaliana Untreated 2 B
SA48049407-CT240125-EB-REP2 neg_RB7_01_44521_modifiedArabidopsis Thaliana Untreated 2 B
SA48049506-CT240125-EB-REP1 neg_RB6_01_44534_modifiedArabidopsis Thaliana Untreated 2 B
SA48049609-CT240125-EB-REP4 pos_RC1_01_44477_modifiedArabidopsis Thaliana Untreated 2 B
SA48049706-CT240125-EB-REP1 pos_RB6_01_44469_modifiedArabidopsis Thaliana Untreated 2 B
SA48049808-CT240125-EB-REP3 pos_RB8_01_44484_modifiedArabidopsis Thaliana Untreated 2 B
SA48049910-CT240125-EB-REP5re pos_RC2_01_44465_modifiedArabidopsis Thaliana Untreated 2 B
SA48050012-CT240125-EC-REP2 pos_RC4_01_44472_modifiedArabidopsis Thaliana Untreated 2 C
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Collection:

Collection ID:CO004295
Collection Summary:Seeds from wild-type Arabidopsis thaliana ecotype Colombia 0 (Col-0) were used for plant growing under no treatment and with biotic and abiotic stresses. 20 mg of seeds were sterilized with 1mL 70% ethanol containing 0.01 % Triton X-100 for 10 minutes. After incubation the solution was removed, and seeds were washed with 96% EtOH and left to dry in sterile conditions. 1 mL of sterile Milli-Q water was added to the tubes containing the seeds and left for stratification at 4°C for 4 days. A. thaliana wild-type Col-0 seeds (20 mg) were grown in 200 mL of sterile liquid Murashige-Skoog (MS) media in a 1-liter Erlenmeyer glass flask on orbital shakers with constant light (120 µE) and temperature (21°C) for a total of 16 days. The shaker speed was kept low (50 rpm) and was gradually increased to 80 rpm after three days, and to 100 rpm after seven days of growth. After 7 days, 50 mL of fresh MS medium was added to the seedlings. On day 14, the media were discarded, and seedlings were washed with 200 mL fresh media (either normal MS media or MS with nutrient deficiency).
Sample Type:Seedlings
Storage Conditions:-80℃

Treatment:

Treatment ID:TR004311
Treatment Summary:The macronutrient deprivation and biotic treatments took place as described previously with some modifications, and lasted for 2 days. In short:• For the untreated plants, 300 mL of fresh MS media were added. The MS media composition was the following: NH4NO3; 1650 mg/L, H3BO3; 6.2 mg/L, CaCl2; 332.2 mg/L, CoCl2*6H2O; 0.025 mg/L, CuSO4*5H2O; 0.025 mg/L, Na2-EDTA; 37.26 mg/L, FeSO4*7H2O; 27.8 mg/L, MgSO4; 180.7 mg/L, MnSO4*H2O; 16.9 mg/L, Na2MoO4*2H2O; 0.25 mg/L, KI; 0.83 mg/L, ZnSO4*7H2O; 8.6 mg/L, KNO3; 1900 mg/L, KH2PO4; 170 mg/L. • For the inorganic Nitrogen starvation, normal MS media were replaced by 300 mL inorganic Nitrogen (- N) deficient media with the following composition: H3BO3; 6.2 mg/L, CaCl2; 332.2 mg/L, CoCl2*6H2O; 0.025 mg/L, CuSO4*5H2O; 0.025 mg/L, Na2-EDTA; 37.26 mg/L, FeSO4*7H2O; 27.8 mg/L, MgSO4; 180.7 mg/L, MnSO4*H2O; 16.9 mg/L, Na2MoO4*2H2O; 0.25 mg/L, KI; 0.83 mg/L, ZnSO4*7H2O; 8.6 mg/L, KNO3; KH2PO4; 170 mg/L, K2SO4; 3278 mg/L. • For the inorganic Phosphorus starvation, normal MS media were replaced by 300 mL inorganic Phosphorus (- P) deficient media with the following composition: NH4NO3; 1650 mg/L, H3BO3; 6.2 mg/L, CaCl2; 332.2 mg/L, CoCl2*6H2O; 0.025 mg/L, CuSO4*5H2O; 0.025 mg/L, Na2-EDTA; 37.26 mg/L, FeSO4*7H2O; 27.8 mg/l, MgSO4; 180.7 mg/L, MnSO4*H2O; 16.9 mg/L, Na2MoO4*2H2O; 0.25 mg/l, KI; 0.83 mg/L, ZnSO4*7H2O; 8.6 mg/L, KNO3; 1900 mg/L. • For the flagellin-related experiments; normal MS media (recipe above) were replaced with 300 mL MS media spiked flagellin-22 in a final concentration of 1 µM flg22. The flagellin-containing media were generated by mixing flagellin 22 (Genscript) with MilliQ water. • For the chitin-related experiments; normal MS media were replaced with 300 mL MS media containing chitin in a final concentration of 0,1 mg/mL. The chitin solution was generated by dissolving chitin (Merck, Darmstadt, Germany) in MilliQ water at 10 mg/mL with gentle shaking together with metal beads for a few hours at 4°C. The resuspended chitin solution was autoclaved at 121°C for 20 min. The solution was centrifuged (3000 rpm at 2 min) to obtain a clear supernatant, which was added to the flask to a concentration of 0.1 mg/mL.

Sample Preparation:

Sampleprep ID:SP004308
Sampleprep Summary:2.3.1 Extraction Protocol A Freeze-dried plant material (100 mg.) was carefully weighed into different 5 mL Eppendorf tubes kept in dry ice and pre-filled with two 3mm diameter metal beads (Bearing Wearhouse). 1.5 mL of Acetonitrile/Methanol (1:1) spiked with 3 μM of internal standard Ampicillin (Merck, Darmstadt, Germany) were added to the tube. The samples were vortexed for 30 seconds and subsequently extracted in a water bath sonicator (Vevor, Shanghai, China) at room temperature for 20 minutes at 60 Hz. The samples were then centrifuged at 4°C at 5000 rpm for 20 minutes. After centrifugation, the supernatant was removed into a 12-mL DURAN glass tube (Hounisen, Skanderborg, Denmark), and the plant material was re-extracted with 1 mL of Acetonitrile/Methanol/Water (4:4:2) spiked with 3 μM of Ampicillin using the same steps. After the second extraction, the supernatant was pooled with the previous one, and a final extraction using 1 mL of Methanol/Water (8:2) spiked with internal standard was conducted similarly. The third supernatant was pooled with the previous two, and the samples were filtered through 0.22 μm PVDF filter pre-conditioned with 300 μl of 50% methanol. After filtration, samples were dried overnight under a mild stream of nitrogen. 2.3.2 Extraction Protocol B Freeze-dried plant material (100 mg.) was carefully weighed into different 5 mL Eppendorf tubes kept in dry-ice and pre-filled with two 3mm diameter metal beads. 1.5 mL of Ethanol/Methanol (1:1) spiked with 3 μM of internal standard Ampicillin (Merck, Darmstadt, Germany) were added to the tube. The samples were vortexed for 30 seconds and subsequently extracted in a water bath sonicator (Vevor, Shanghai, China) at room temperature for 20 minutes at 60 Hz. The samples were then centrifuged at 4°C at 5000 rpm for 20 minutes. After centrifugation, the supernatant was removed into a 12-mL DURAN glass tube (Hounisen, Skanderborg, Denmark), and the plant material was re-extracted with 1 mL of Ethanol/Methanol/Water (4:4:2) spiked with 3 μM of Ampicillin using the same steps. After the second extraction, the supernatant was pooled with the previous one, and a final extraction using 1 mL of Methanol/Water (8:2) spiked with internal standard was conducted similarly. The third supernatant was pooled with the previous two and the samples were filtered through 0.22 μm PVDF filter pre-conditioned with 300 μl of 50% methanol. After filtration, samples were dried overnight under a mild stream of nitrogen and stored at -80°C. 2.3.3 Extraction Protocol C Freeze-dried plant material (100 mg.) was carefully weighed into different 5 mL Eppendorf tubes kept in dry-ice and pre-filled with two 3mm diameter metal beads. 1.5 mL of Methanol/Water (8:2) spiked with 3 μM of internal standard were added to the tube. The samples were vortexed for 30 seconds and subsequently extracted in a water bath sonicator (Vevor, Shanghai, China) at room temperature for 20 minutes at 60 Hz. The samples were then centrifuged at 4°C at 5000 rpm for 20 minutes. After centrifugation, the supernatant was removed into a 12-mL DURAN glass tube (Hounisen, Skanderborg, Denmark), and the plant material was re-extracted with 1 mL of the same solvent using the exact same steps. After the second extraction the supernatant was pooled with the previous one and a final re-extraction using 1 mL was conducted similarly. The third supernatant was pooled with the previous two, and the samples were filtered through 0.22 μm PVDF filter pre-conditioned with 300 μL of 50% methanol. After filtration, samples were dried overnight under a mild stream of nitrogen and stored at -80°C. 2.3.4 Extraction Protocol D Freeze-dried plant material (100 mg.) was carefully weighed into different 5 mL Eppendorf tubes kept in dry-ice and pre-filled with two 3mm diameter metal beads. 1.5 mL of Dichloromethane/Methanol (3:1) spiked with 3 μM of internal standard were added to the tube. The samples were vortexed for 30 seconds and subsequently extracted in a water bath sonicator (Vevor, Shanghai, China) at room temperature for 20 minutes at 60 Hz. The samples were then centrifuged at 4 °C at 5000 rpm for 20 minutes. After centrifugation, the supernatant was removed into a 12-mL DURAN glass tube (Hounisen, Skanderborg, Denmark), and the plant material was re-extracted with 1 mL of the same solvent using the exact same steps. After the second extraction the supernatant was pooled with the previous one and a final re-extraction using 1 mL of Methanol/Water (8:2) spiked with 3 μM of internal standard was performed. The third supernatant was pooled with the previous two into the 12 mL glass tubes and the samples were filtered through 0.22 μm PVDF filter pre-conditioned with 300 μl of 50% methanol followed by the addition of 1 mL of milliQ water to induce phase separation. The tube was left to stand in room temperature for 5 minutes and was subsequently centrifuged at 3000 g at 4°C for 20 minutes. After centrifugation, the upper MeOH-H2O was removed carefully in a new 12-mL glass tube, while the lower DCM (Dichloromethane) phase was re-extracted with 1.5 mL of water with the same procedure as the previous step. After the second phase separation, the upper phase containing the secondary metabolites was pooled with the previous upper phase, and samples were dried overnight under a mild stream of nitrogen and stored at -80°C.

Chromatography:

Chromatography ID:CH005230
Chromatography Summary:Pellets were resuspended in 2 mL of 50% MeOH. After 20-fold dilution samples were filtered with 0.22 μm syringe filters (PVDF) and subjected to analysis. Separation was achieved on a Dionex UltiMate 3000 Quaternary Rapid Separation UHPLC+ focused system (Thermo Fisher Scientific, Germering, Germany) equipped with a ZORBAX Eclipse 1.8 μm XDB-C18 column (100 × 3 mm, 1.8 μm, Agilent). For eluting 0.05% (v/v) formic acid in H2O and 0.05% (v/v) formic acid in MeCN were employed as mobile phases A and B, respectively. Gradient conditions were as follows: 0.0−1.0 min 3% B; 1.0−14.0 min 3−20% B; 14.0−20.0 min 20−45% B, 20.0−24.5 min 45−100% B, 24.5−26.5 min 100% B, 26.5−26.55 min 100−3% B, and 26.55−30.0 min 3% B. The flow rate of the mobile phase was 400 μL/min and injection volume were set at 20 μL. The column temperature was maintained at 40°C. UV chromatograms were acquired at 229, 260, 310, and 345 nm. The UHPLC was coupled to a Compact micro-TOF-Q mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive or negative ionization mode. The ion spray voltage was maintained at 3750 in positive and -4000 V in negative ionization mode. The dry temperature was set to 275 (positive) and 325°C (negative), and the dry gas flow was set to 8 L/min. Nitrogen was used as the dry gas, nebulizing gas, and collision gas. The nebulizing gas was set to 2.5 bar and collision energy to 15 eV. HRESIMS and MS/MS spectra were acquired in an m/z range from 50 to 1200 amu at a sampling rate of 2 Hz. Sodium formate clusters were used for mass calibration. All files were automatically calibrated by postprocessing. All acquired chromatograms and spectra were analyzed with Compass DataAnalysis v.4.3.0 (Bruker, Bremen, Germany).
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Agilent ZORBAX Eclipse XDB C-18 (100 × 3 mm, 1.8 μm)
Column Temperature:40°C
Flow Gradient:0.0−1.0 min 3% B; 1.0−14.0 min 3−20% B; 14.0−20.0 min 20−45% B, 20.0−24.5 min 45−100% B, 24.5−26.5 min 100% B, 26.5−26.55 min 100−3% B, and 26.55−30.0 min 3% B
Flow Rate:400 μL/min
Solvent A:100% Water; 0.05% Formic acid
Solvent B:100% Acetonitrile; 0.05% Formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH005231
Chromatography Summary:Pellets were resuspended in 2 mL of 50% MeOH. After 20-fold dilution samples were filtered with 0.22 μm syringe filters (PVDF) and subjected to analysis. Separation was achieved on a Dionex UltiMate 3000 Quaternary Rapid Separation UHPLC+ focused system (Thermo Fisher Scientific, Germering, Germany) equipped with a ZORBAX Eclipse 1.8 μm XDB-C18 column (100 × 3 mm, 1.8 μm, Agilent). For eluting 0.05% (v/v) formic acid in H2O and 0.05% (v/v) formic acid in MeCN were employed as mobile phases A and B, respectively. Gradient conditions were as follows: 0.0−1.0 min 3% B; 1.0−14.0 min 3−20% B; 14.0−20.0 min 20−45% B, 20.0−24.5 min 45−100% B, 24.5−26.5 min 100% B, 26.5−26.55 min 100−3% B, and 26.55−30.0 min 3% B. The flow rate of the mobile phase was 400 μL/min and injection volume were set at 20 μL. The column temperature was maintained at 40°C. UV chromatograms were acquired at 229, 260, 310, and 345 nm. The UHPLC was coupled to a Compact micro-TOF-Q mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive or negative ionization mode. The ion spray voltage was maintained at 3750 in positive and -4000 V in negative ionization mode. The dry temperature was set to 275 (positive) and 325°C (negative), and the dry gas flow was set to 8 L/min. Nitrogen was used as the dry gas, nebulizing gas, and collision gas. The nebulizing gas was set to 2.5 bar and collision energy to 15 eV. HRESIMS and MS/MS spectra were acquired in an m/z range from 50 to 1200 amu at a sampling rate of 2 Hz. Sodium formate clusters were used for mass calibration. All files were automatically calibrated by postprocessing. All acquired chromatograms and spectra were analyzed with Compass DataAnalysis v.4.3.0 (Bruker, Bremen, Germany).
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Agilent ZORBAX Eclipse XDB C-18 (100 × 3 mm, 1.8 μm)
Column Temperature:40°C
Flow Gradient:0.0−1.0 min 3% B; 1.0−14.0 min 3−20% B; 14.0−20.0 min 20−45% B, 20.0−24.5 min 45−100% B, 24.5−26.5 min 100% B, 26.5−26.55 min 100−3% B, and 26.55−30.0 min 3% B
Flow Rate:400 μL/min
Solvent A:100% Water; 0.05% Formic acid
Solvent B:100% Acetonitrile; 0.05% Formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH005232
Chromatography Summary:The metabolite residue was dissolved in 3 mL of 80% MeOH containing 2.5 μM lidocaine, 2.5 μM 10-camphour sulfonic acid, and 10.4 ppm acetic acid. Then, 150 μL of the re-extraction was loaded and eluted with an HLB μElution plate (Waters) that had been conditioned with 200 μL of MeOH and equilibrated with 200 μL of 80% MeOH containing 10.4 ppm acetic acid. The extracts (1 μL) were subsequently analyzed using LC-QTOF-MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). Analytical conditions were as follows LC: column, Acquity bridged ethyl hybrid (BEH) C18 (1.7 μm, 2.1 mm * 100 mm, Waters); solvent system, solvent A (water including 0.1% [v/v] formic acid ) and solvent B (acetonitrile including 0.1% [v/v] formic acid); gradient program, 99.5%A/0.5%B at 0 min, 99.5%A/0.5%B at 0.1 min, 20%A/80%B at 10 min, 0.5%A/99.5%B at 10.1 min, 0.5%A/99.5%B at 12.0 min, 99.5%A/0.5%B at 12.1 min and 99.5%A/0.5%B at 15.0 min; flow rate, 0.3 mL/min at 0 min, 0.3 mL/min at 10 min, 0.4 mL/min at 10.1 min, 0.4 mL/min at 14.4 min and 0.3 mL/min at 14.5 min; column temperature, 40 °C; MS detection: polarity, positive/negative; capillary voltage, +3.00 kV (positive)/-2.75 kV (negative); cone voltage, 25.0 V; source temperature, 120 °C; desolvation temperature, 450°C; cone gas flow, 50 l/h; desolvation gas flow, 800 l/h; collision energy, 6 V; mass range, m/z 50‒1500; scan duration, 0.1 sec; interscan delay, 0.014 sec; data acquisition, centroid mode; Lockspray (Leucine enkephalin); scan duration, 1.0 sec; interscan delay, 0.1 sec. MS/MS data was acquired in the ramp mode as the following analytical conditions: (1) MS: polarity, positive/negative; mass range, m/z 50–1500; scan duration, 0.1 sec; inter-scan delay, 0.014 sec; data acquisition, centroid mode and (2) MS/MS: polarity, positive/negative; mass range, m/z 50–1500; scan duration, 0.02 or 0.1 sec; inter-scan delay, 0.014 sec; data acquisition, centroid mode; collision energy, ramped from 10 to 50 V. In this mode, MS/MS spectra of the top 10 ions (> 1000 counts) in an MS scan were automatically obtained. If the ion intensity was less than 1000, MS/MS data acquisition was not performed and moved to of next top 10 ions.
Instrument Name:Waters Acquity
Column Name:Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 μm)
Column Temperature:40°C
Flow Gradient:99.5%A/0.5%B at 0 min, 99.5%A/0.5%B at 0.1 min, 20%A/80%B at 10 min, 0.5%A/99.5%B at 10.1 min, 0.5%A/99.5%B at 12.0 min, 99.5%A/0.5%B at 12.1 min and 99.5%A/0.5%B at 15.0 min
Flow Rate:flow rate, 0.3 mL/min at 0 min, 0.3 mL/min at 10 min, 0.4 mL/min at 10.1 min, 0.4 mL/min at 14.4 min and 0.3 mL/min at 14.5 min
Solvent A:100% Water; 0.1% Formic acid
Solvent B:100% Acetonitrile; 0.1% Formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH005233
Chromatography Summary:The metabolite residue was dissolved in 3 mL of 80% MeOH containing 2.5 μM lidocaine, 2.5 μM 10-camphour sulfonic acid, and 10.4 ppm acetic acid. Then, 150 μL of the re-extraction was loaded and eluted with an HLB μElution plate (Waters) that had been conditioned with 200 μL of MeOH and equilibrated with 200 μL of 80% MeOH containing 10.4 ppm acetic acid. The extracts (1 μL) were subsequently analyzed using LC-QTOF-MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). Analytical conditions were as follows LC: column, Acquity bridged ethyl hybrid (BEH) C18 (1.7 μm, 2.1 mm * 100 mm, Waters); solvent system, solvent A (water including 0.1% [v/v] formic acid ) and solvent B (acetonitrile including 0.1% [v/v] formic acid); gradient program, 99.5%A/0.5%B at 0 min, 99.5%A/0.5%B at 0.1 min, 20%A/80%B at 10 min, 0.5%A/99.5%B at 10.1 min, 0.5%A/99.5%B at 12.0 min, 99.5%A/0.5%B at 12.1 min and 99.5%A/0.5%B at 15.0 min; flow rate, 0.3 mL/min at 0 min, 0.3 mL/min at 10 min, 0.4 mL/min at 10.1 min, 0.4 mL/min at 14.4 min and 0.3 mL/min at 14.5 min; column temperature, 40 °C; MS detection: polarity, positive/negative; capillary voltage, +3.00 kV (positive)/-2.75 kV (negative); cone voltage, 25.0 V; source temperature, 120 °C; desolvation temperature, 450°C; cone gas flow, 50 l/h; desolvation gas flow, 800 l/h; collision energy, 6 V; mass range, m/z 50‒1500; scan duration, 0.1 sec; interscan delay, 0.014 sec; data acquisition, centroid mode; Lockspray (Leucine enkephalin); scan duration, 1.0 sec; interscan delay, 0.1 sec. MS/MS data was acquired in the ramp mode as the following analytical conditions: (1) MS: polarity, positive/negative; mass range, m/z 50–1500; scan duration, 0.1 sec; inter-scan delay, 0.014 sec; data acquisition, centroid mode and (2) MS/MS: polarity, positive/negative; mass range, m/z 50–1500; scan duration, 0.02 or 0.1 sec; inter-scan delay, 0.014 sec; data acquisition, centroid mode; collision energy, ramped from 10 to 50 V. In this mode, MS/MS spectra of the top 10 ions (> 1000 counts) in an MS scan were automatically obtained. If the ion intensity was less than 1000, MS/MS data acquisition was not performed and moved to of next top 10 ions.
Instrument Name:Waters Acquity
Column Name:Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 μm)
Column Temperature:40°C
Flow Gradient:99.5%A/0.5%B at 0 min, 99.5%A/0.5%B at 0.1 min, 20%A/80%B at 10 min, 0.5%A/99.5%B at 10.1 min, 0.5%A/99.5%B at 12.0 min, 99.5%A/0.5%B at 12.1 min and 99.5%A/0.5%B at 15.0 min
Flow Rate:flow rate, 0.3 mL/min at 0 min, 0.3 mL/min at 10 min, 0.4 mL/min at 10.1 min, 0.4 mL/min at 14.4 min and 0.3 mL/min at 14.5 min
Solvent A:100% Water; 0.1% Formic acid
Solvent B:100% Acetonitrile; 0.1% Formic acid
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN006883
Analysis Type:MS
Chromatography ID:CH005230
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006883_Results.txt
Units:dalton
  
Analysis ID:AN006884
Analysis Type:MS
Chromatography ID:CH005230
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006884_Results.txt
Units:dalton
  
Analysis ID:AN006885
Analysis Type:MS
Chromatography ID:CH005230
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006885_Results.txt
Units:dalton
  
Analysis ID:AN006886
Analysis Type:MS
Chromatography ID:CH005230
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006886_Results.txt
Units:dalton
  
Analysis ID:AN006887
Analysis Type:MS
Chromatography ID:CH005231
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006887_Results.txt
Units:dalton
  
Analysis ID:AN006888
Analysis Type:MS
Chromatography ID:CH005231
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006888_Results.txt
Units:dalton
  
Analysis ID:AN006889
Analysis Type:MS
Chromatography ID:CH005231
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006889_Results.txt
Units:dalton
  
Analysis ID:AN006890
Analysis Type:MS
Chromatography ID:CH005231
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006890_Results.txt
Units:dalton
  
Analysis ID:AN006891
Analysis Type:MS
Chromatography ID:CH005232
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006891_Results.txt
Units:dalton
  
Analysis ID:AN006892
Analysis Type:MS
Chromatography ID:CH005233
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004151_AN006892_Results.txt
Units:dalton
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