Summary of Study ST002778

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 PR001733. The data can be accessed directly via it's Project DOI: 10.21228/M8PX3J This work is supported by NIH grant, U2C- DK119886.

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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 IDST002778
Study TitleCell Lineage-Guided Microanalytical Mass Spectrometry Reveals Increased Energy Metabolism and Reactive Oxygen Species in the Vertebrate Organizer
Study SummaryWe performed targeted metabolomic analysis on the Spemann-Mangold Organizer (SMO) tissue in the frog (Xenopus laevis) and the remainder of dissected embryos (RE). Metabolites were extracted from the dissected tissues, reconstituted, and analyzed using liquid chromatography (LC) electrospray ionization (ESI) mass spectrometry (MS). The targeted metabolite measurements were performed on a trapped ion mobility time-of-flight mass spectrometer (timsTOF PRO, Bruker).
Institute
University of Maryland
DepartmentChemistry & Biochemistry
Last NameNemes
First NamePeter
Address8051 Regents Drive, College Park, MD 20742, USA
Emailnemes@umd.edu
Phone3014050373
Submit Date2023-07-03
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2024-01-10
Release Version1
Peter Nemes Peter Nemes
https://dx.doi.org/10.21228/M8PX3J
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001733
Project DOI:doi: 10.21228/M8PX3J
Project Title:Cell Lineage-Guided Microanalytical Mass Spectrometry Reveals Increased Energy Metabolism and Reactive Oxygen Species in the Vertebrate Organizer
Project Summary:We performed targeted metabolomic analysis on the Spemann-Mangold Organizer (SMO) tissue in the frog (Xenopus laevis) and the remainder of dissected embryos (RE). The goal of this study is to quantify a panel of targeted metabolite intermediates from glycolysis, phosphate energy pool, and mitochondrial activity including the TCA cycle. Metabolites were extracted from the dissected tissues, reconstituted, and analyzed using liquid chromatography (LC) electrospray ionization (ESI) mass spectrometry (MS). The targeted metabolite measurements were performed on a trapped ion mobility time-of-flight mass spectrometer (timsTOF PRO, Bruker). Targeted MS assays on the metabolite intermediates produced downstream, complemented by classical fluorescence-based metabolite assays when available, revealed local oxidative stress and enrichment of reactive oxygen species (ROS) in the SMO.
Institute:University of Maryland
Department:Chemistry & Biochemistry
Last Name:Nemes
First Name:Peter
Address:8051 Regents Drive, College Park, MD 20742, USA
Email:nemes@umd.edu
Phone:301-405-0373
Funding Source:National Institutes of Health under Award no. 1R35GM124755
Contributors:Aparna B. Baxi, Jie Li, Vi M. Quach, and Peter Nemes

Subject:

Subject ID:SU002885
Subject Type:Amphibian
Subject Species:Xenopus laevis
Taxonomy ID:8355

Factors:

Subject type: Amphibian; Subject species: Xenopus laevis (Factor headings shown in green)

mb_sample_id local_sample_id Sample type
SA2973222021-01-31 JL12 SO WE 2 MRM TR1_1-ERE
SA2973232021-09-10 JL54 SO1 3x dilution GSH GSSG MRM TR3_1-BRE
SA2973242021-01-31 JL06 SO WE 1 MRM TR2_1-ERE
SA2973252021-01-31 JL13 SO WE 2 MRM TR2_1-ERE
SA2973262021-01-31 JL07 SO WE 1 MRM TR3_1-ERE
SA2973272021-01-31 JL14 SO WE 2 MRM TR3_1-ERE
SA2973282021-01-31 JL19 SO WE 3 MRM TR1_1-ERE
SA2973292021-09-10 JL52 SO1 3x dilution GSH GSSG MRM TR1_1-BRE
SA2973302021-09-10 JL53 SO1 3x dilution GSH GSSG MRM TR2_1-BRE
SA2973312021-01-31 JL05 SO WE 1 MRM TR1_1-ERE
SA2973322021-09-10 JL60 SO2 3x dilution GSH GSSG MRM TR1_1-BRE
SA2973332021-02-01 JL55 SO WE 8 MRM LM TR2_1-ERE
SA2973342021-02-01 JL54 SO WE 8 MRM LM TR1_1-ERE
SA2973352021-09-10 JL68 SO3 3x dilution GSH GSSG MRM TR1_1-BRE
SA2973362021-09-10 JL62 SO2 3x dilution GSH GSSG MRM TR3_1-BRE
SA2973372021-02-01 JL61 SO WE 9 MRM LM TR1_1-FRE
SA2973382021-09-10 JL61 SO2 3x dilution GSH GSSG MRM TR2_1-BRE
SA2973392021-02-01 JL63 SO WE 9 MRM LM TR3_1-FRE
SA2973402021-02-01 JL62 SO WE 9 MRM LM TR2_1-FRE
SA2973412021-01-31 JL20 SO WE 3 MRM TR2_1-ERE
SA2973422021-01-31 JL21 SO WE 3 MRM TR3_1-ERE
SA2973432021-01-31 JL42 SO WE 6 MRM TR3_1-ERE
SA2973442021-01-31 JL41 SO WE 6 MRM TR2_1-ERE
SA2973452021-01-31 JL40 SO WE 6 MRM TR1_1-ERE
SA2973462021-01-31 JL55 SO WE 8 MRM TR2_1-ERE
SA2973472021-01-31 JL54 SO WE 8 MRM TR1_1-ERE
SA2973482021-01-31 JL49 SO WE 7 MRM TR3_1-ERE
SA2973492021-01-31 JL48 SO WE 7 MRM TR2_1-ERE
SA2973502021-01-31 JL47 SO WE 7 MRM TR1_1-ERE
SA2973512021-01-31 JL56 SO WE 8 MRM TR3_1-ERE
SA2973522021-01-31 JL35 SO WE 5 MRM TR3_1-ERE
SA2973532021-01-31 JL27 SO WE 4 MRM TR2_1-ERE
SA2973542021-01-31 JL26 SO WE 4 MRM TR1_1-ERE
SA2973552021-01-31 JL63 SO WE 9 MRM TR3_1-FRE
SA2973562021-01-31 JL28 SO WE 4 MRM TR3_1-ERE
SA2973572021-01-31 JL62 SO WE 9 MRM TR2_1-FRE
SA2973582021-01-31 JL34 SO WE 5 MRM TR2_1-ERE
SA2973592021-01-31 JL33 SO WE 5 MRM TR1_1-ERE
SA2973602021-01-31 JL61 SO WE 9 MRM TR1_1-FRE
SA2973612021-09-10 JL69 SO3 3x dilution GSH GSSG MRM TR2_1-BRE
SA2973622021-02-01 JL56 SO WE 8 MRM LM TR3_1-ERE
SA2973632021-02-01 JL28 SO WE 4 MRM LM TR3_1-ERE
SA2973642021-02-01 JL49 SO WE 7 MRM LM TR3_1-ERE
SA2973652021-02-01 JL27 SO WE 4 MRM LM TR2_1-ERE
SA2973662021-09-10 JL78 SO4 3x dilution GSH GSSG MRM TR3_1-BRE
SA2973672021-02-01 JL33 SO WE 5 MRM LM TR1_1-ERE
SA2973682021-02-01 JL13 SO WE 2 MRM LM TR2_1-ERE
SA2973692021-02-01 JL34 SO WE 5 MRM LM TR2_1-ERE
SA2973702021-02-01 JL26 SO WE 4 MRM LM TR1_1-ERE
SA2973712021-02-01 JL14 SO WE 2 MRM LM TR3_1-ERE
SA2973722021-02-01 JL19 SO WE 3 MRM LM TR1_1-ERE
SA2973732021-09-10 JL86 SO5 3x dilution GSH GSSG MRM TR3_1-CRE
SA2973742021-02-01 JL20 SO WE 3 MRM LM TR2_1-ERE
SA2973752021-02-01 JL21 SO WE 3 MRM LM TR3_1-ERE
SA2973762021-09-10 JL84 SO5 3x dilution GSH GSSG MRM TR1_1-CRE
SA2973772021-09-10 JL85 SO5 3x dilution GSH GSSG MRM TR2_1-CRE
SA2973782021-02-01 JL35 SO WE 5 MRM LM TR3_1-ERE
SA2973792021-02-01 JL12 SO WE 2 MRM LM TR1_1-ERE
SA2973802021-02-01 JL06 SO WE 1 MRM LM TR2_1-ERE
SA2973812021-02-01 JL07 SO WE 1 MRM LM TR3_1-ERE
SA2973822021-09-10 JL77 SO4 3x dilution GSH GSSG MRM TR2_1-BRE
SA2973832021-02-01 JL05 SO WE 1 MRM LM TR1_1-ERE
SA2973842021-09-10 JL70 SO3 3x dilution GSH GSSG MRM TR3_1-BRE
SA2973852021-02-01 JL48 SO WE 7 MRM LM TR2_1-ERE
SA2973862021-09-10 JL94 SO6 3x dilution GSH GSSG MRM TR3_1-CRE
SA2973872021-02-01 JL47 SO WE 7 MRM LM TR1_1-ERE
SA2973882021-02-01 JL42 SO WE 6 MRM LM TR3_1-ERE
SA2973892021-09-10 JL93 SO6 3x dilution GSH GSSG MRM TR2_1-CRE
SA2973902021-02-01 JL41 SO WE 6 MRM LM TR2_1-ERE
SA2973912021-09-10 JL92 SO6 3x dilution GSH GSSG MRM TR1_1-CRE
SA2973922021-09-10 JL76 SO4 3x dilution GSH GSSG MRM TR1_1-BRE
SA2973932021-02-01 JL40 SO WE 6 MRM LM TR1_1-ERE
SA2973942021-01-31 JL60 SO9 MRM TR3_1-DSMO
SA2973952021-09-10 JL90 SOWE6 3x dilution GSH GSSG MRM TR3_1-CSMO
SA2973962021-01-31 JL58 SO9 MRM TR1_1-DSMO
SA2973972021-01-31 JL59 SO9 MRM TR2_1-DSMO
SA2973982021-01-31 JL53 SO8 MRM TR3_1-CSMO
SA2973992021-09-10 JL89 SOWE6 3x dilution GSH GSSG MRM TR2_1-CSMO
SA2974002021-09-10 JL88 SOWE6 3x dilution GSH GSSG MRM TR1_1-CSMO
SA2974012021-09-10 JL50 SOWE1 3x dilution GSH GSSG MRM TR3_1-BSMO
SA2974022021-09-10 JL72 SOWE4 3x dilution GSH GSSG MRM TR1_1-BSMO
SA2974032021-09-10 JL73 SOWE4 3x dilution GSH GSSG MRM TR2_1-BSMO
SA2974042021-09-10 JL64 SOWE3 3x dilution GSH GSSG MRM TR1_1-BSMO
SA2974052021-09-10 JL65 SOWE3 3x dilution GSH GSSG MRM TR2_1-BSMO
SA2974062021-09-10 JL66 SOWE3 3x dilution GSH GSSG MRM TR3_1-BSMO
SA2974072021-01-31 JL52 SO8 MRM TR2_1-CSMO
SA2974082021-09-10 JL74 SOWE4 3x dilution GSH GSSG MRM TR3_1-BSMO
SA2974092021-09-10 JL58 SOWE2 3x dilution GSH GSSG MRM TR3_1-BSMO
SA2974102021-09-10 JL82 SOWE5 3x dilution GSH GSSG MRM TR3_1-CSMO
SA2974112021-09-10 JL49 SOWE1 3x dilution GSH GSSG MRM TR2_1-BSMO
SA2974122021-09-10 JL81 SOWE5 3x dilution GSH GSSG MRM TR2_1-CSMO
SA2974132021-09-10 JL80 SOWE5 3x dilution GSH GSSG MRM TR1_1-CSMO
SA2974142021-09-10 JL57 SOWE2 3x dilution GSH GSSG MRM TR2_1-BSMO
SA2974152021-09-10 JL56 SOWE2 3x dilution GSH GSSG MRM TR1_1-BSMO
SA2974162021-09-10 JL48 SOWE1 3x dilution GSH GSSG MRM TR1_1-BSMO
SA2974172021-01-31 JL23 SO4 MRM TR1_1-CSMO
SA2974182021-02-01 JL38 SO6 MRM LM TR2_1-CSMO
SA2974192021-02-01 JL39 SO6 MRM LM TR3_1-CSMO
SA2974202021-02-01 JL37 SO6 MRM LM TR1_1-CSMO
SA2974212021-02-01 JL32 SO5 MRM LM TR3_1-CSMO
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Collection:

Collection ID:CO002878
Collection Summary:The Spemann-Mangold Organizer (SMO) was lineage-traced under epifluorescence and isolated in a 2% (w/v) agarose-coated Petri dish containing 50% SS. The dissected SMO tissues and the remainder embryo (RE) were collected in LoBind Eppendorf tubes separately, and processed using our established protocols for metabolomic analysis. For MS-based metabolomics of the TCA cycle and glycolysis, 10 dissections of SMO and RE were pooled as 1 BR. For targeted quantification of reduced and oxidized glutathione, 20 tissues of SMO and RE were dissected and pooled as one BR. The dissected tissues were stored in LC-MS-grade methanol at −80 °C until sample processing.
Sample Type:Dissected organizer from Xenopus laevis

Treatment:

Treatment ID:TR002894
Treatment Summary:NA

Sample Preparation:

Sampleprep ID:SP002891
Sampleprep Summary:The supernatant was transferred to a new 1.5 mL Eppendorf vial, vacuum-dried at 20 °C, and reconstituted in 45 uL of LC-MS grade water. For GSH/GSSG analysis, the supernatant was reconstituted in 45 uL of 50% ACN. The supernatant was centrifuged at 13,000 g for 10 min at 4 °C, then stored at -80 °C until LC-MS analysis.
Sampleprep Protocol Filename:Baxi_2023_Organizer_Work_MeabolomicsWorkBench_Description_2023-07-02.docx

Combined analysis:

Analysis ID AN004522 AN004523 AN004524
Analysis type MS MS MS
Chromatography type Ion exchange Ion exchange HILIC
Chromatography system Waters ACQUITY I-Class Waters Acquity I-Class Waters Acquity I-Class
Column Waters Atlantis Premier BEH C18 AX (100 x 2.1mm, 1.7um) Waters Atlantis Premier BEH C18 AX (100 x 2.1mm, 1.7um) ACQUITY UPLC BEH amide (100 x 1mm, 1.7um)
MS Type ESI ESI ESI
MS instrument type QTOF QTOF QTOF
MS instrument name Bruker timsTOF PRO Bruker timsTOF PRO Bruker timsTOF PRO
Ion Mode NEGATIVE NEGATIVE POSITIVE
Units Counts Counts Counts

Chromatography:

Chromatography ID:CH003396
Chromatography Summary:A 2.5 µL of the metabolite extract was loaded onto a reversed-phase LC column featuring anion-exchange chemistry (Atlantis PREMIER BEH C18 AX Column, Waters, 1.7 µm, 2.1 × 100 mm) and separated at 500 uL/min in a micro-flow LC system (ACQUITY I-Class, Waters) using a gradient of buffer A (100% ACN) and buffer B (95% water, 5% ACN with 1 mM ammonium acetate; pH 7.8) as follows: 5% was held 0–¬0.5 min, then linearly increased to 90% over 9.5 min, and held at 90% for 3 min. To recover and recondition the analytical column before the next sample injection, solvent B was then decreased to 5% over 2 min and the column was rinsed for 7 min. The column temperature was set to 30 °C.
Methods Filename:Baxi_2023_Organizer_Work_MeabolomicsWorkBench_Description_2023-07-02.docx
Instrument Name:Waters ACQUITY I-Class
Column Name:Waters Atlantis Premier BEH C18 AX (100 x 2.1mm, 1.7um)
Column Temperature:30
Flow Gradient:5% B was held 0–0.5 min, then linearly increased to 90% over 9.5 min, and held at 90% for 3 min
Flow Rate:500 uL/min
Solvent A:100% ACN
Solvent B:95% water/5% acetonitrile; 1 mM ammonium acetate; pH 7.8
Chromatography Type:Ion exchange
  
Chromatography ID:CH003397
Chromatography Summary:A 2.5 µL of the metabolite extract was loaded onto a reversed-phase LC column featuring anion-exchange chemistry (Atlantis PREMIER BEH C18 AX Column, Waters, 1.7 µm, 2.1 × 100 mm) and separated at 500 uL/min in a micro-flow LC system (ACQUITY I-Class, Waters) using a gradient of buffer A (100% ACN) and buffer B (95% water, 5% ACN with 1 mM ammonium acetate; pH 7.8) as follows: 5% was held 0–¬0.5 min, then linearly increased to 90% over 9.5 min, and held at 90% for 3 min. To recover and recondition the analytical column before the next sample injection, solvent B was then decreased to 5% over 2 min and the column was rinsed for 7 min. The column temperature was set to 30 °C.
Methods Filename:Baxi_2023_Organizer_Work_MeabolomicsWorkBench_Description_2023-07-02.docx
Instrument Name:Waters Acquity I-Class
Column Name:Waters Atlantis Premier BEH C18 AX (100 x 2.1mm, 1.7um)
Column Temperature:30
Flow Gradient:5% B was held 0–0.5 min, then linearly increased to 90% over 9.5 min, and held at 90% for 3 min
Flow Rate:500 uL/min
Solvent A:100% ACN
Solvent B:95% water/5% acetonitrile; 1 mM ammonium acetate; pH 7.8
Chromatography Type:Ion exchange
  
Chromatography ID:CH003398
Chromatography Summary:A 2-µL volume of the metabolite extract was loaded onto an ACQUITY UPLC BEH amide column (130 A, 1.7 µm, 1 mm × 100 mm) and separated at 45 ℃ using a mobile phase delivered at 130 uL/min in a micro-LC system (Waters ACQUITY I-Class). The separation was performed in buffer A (water containing 0.1% v/v FA) with a gradient of buffer B (100% ACN with 0.1% v/v FA) as follows: 1% A for 0.05 min, then ramped to 80% A over 3.20 min, held at 80% A for 2 min, ramped to 1% A in 1.75 min. To recover and condition the analytical column for the next sample injection, buffer A was held at 1% for 7 min.
Methods Filename:Baxi_2023_Organizer_Work_MeabolomicsWorkBench_Description_2023-07-02.docx
Instrument Name:Waters Acquity I-Class
Column Name:ACQUITY UPLC BEH amide (100 x 1mm, 1.7um)
Column Temperature:45
Flow Gradient:1% A for 0.05 min, then ramped to 80% A over 3.20 min, held at 80% A for 2 min, ramped to 1% A in 1.75 min
Flow Rate:130 uL/min
Solvent A:water containing 0.1% v/v FA
Solvent B:100% acetonitrile; 0.1% v/v FA
Chromatography Type:HILIC

MS:

MS ID:MS004269
Analysis ID:AN004522
Instrument Name:Bruker timsTOF PRO
Instrument Type:QTOF
MS Type:ESI
MS Comments:The metabolites were ionized in a micro-flow electrospray source equipped with an ionBooster-ESI device with the following settings: end plate, 400 V; capillary voltage, 1,000 V; charge voltage, 300 V; vaporizer temperature, 240 °C; sheath gas, 1.5 L/min; nebulizer, 4.1 bar; dry gas, 2.6 L/min; dry temperature, 200 °C. The ions were detected in the negative ionization mode using the following settings: survey (MS1) low- and middle-mass range, m/z 20–300 and m/z 50–1,300, respectively; spectral acquisition rate, 2 Hz. A list of metabolite ions was targeted using multiple reaction monitoring. Ions were isolated with 4-Da window (± 2 Da) for collision-induced dissociation (CID). The low-mass range (m/z 20–300) employed the following optimization for collision energy and m/z tuning: 12 eV at 115.0026; 12 eV at 145.0132; 12 eV at 168.9897; 12 eV at 184.9846; 10 eV at 191.0186.
Ion Mode:NEGATIVE
Analysis Protocol File:Baxi_2023_Organizer_Work_MeabolomicsWorkBench_Description_2023-07-02.docx
  
MS ID:MS004270
Analysis ID:AN004523
Instrument Name:Bruker timsTOF PRO
Instrument Type:QTOF
MS Type:ESI
MS Comments:The metabolites were ionized in a micro-flow electrospray source equipped with an ionBooster-ESI device with the following settings: end plate, 400 V; capillary voltage, 1,000 V; charge voltage, 300 V; vaporizer temperature, 240 °C; sheath gas, 1.5 L/min; nebulizer, 4.1 bar; dry gas, 2.6 L/min; dry temperature, 200 °C. The ions were detected in the negative ionization mode using the following settings: survey (MS1) low- and middle-mass range, m/z 20–300 and m/z 50–1,300, respectively; spectral acquisition rate, 2 Hz. A list of metabolite ions was targeted using multiple reaction monitoring. Ions were isolated with 4-Da window (± 2 Da) for collision-induced dissociation (CID). The middle-mass range (m/z 50–1,300) employed the following optimization for collision energy and m/z tuning: 15 eV at 338.9877; 18 eV at 346.0553; 25 eV at 426.0216; 20 eV at 505.9879.
Ion Mode:NEGATIVE
Analysis Protocol File:Baxi_2023_Organizer_Work_MeabolomicsWorkBench_Description_2023-07-02.docx
  
MS ID:MS004271
Analysis ID:AN004524
Instrument Name:Bruker timsTOF PRO
Instrument Type:QTOF
MS Type:ESI
MS Comments:The GSH and GSSG ion signals were measured in the positive ion mode and detected on the timsTOF PRO (Bruker) mass spectrometer with the following parameters: mass range, m/z 100–800; spectral acquisition rate, 4 Hz; scan mode, MRM (GSH: m/z 308.0911, charge state +1, retention time 2.5 min, width 4 Da (+/– 2 Da), and collision energy 26 eV); and GSSG (m/z 307.0833, charge state +2, retention time 2.9 min, width 4 Da (+/– 2 Da), collision energy 20 eV).
Ion Mode:POSITIVE
Analysis Protocol File:Baxi_2023_Organizer_Work_MeabolomicsWorkBench_Description_2023-07-02.docx
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