Return to study ST002778 main page
MB Sample ID: SA297371
| Local Sample ID: | 2021-02-01 JL14 SO WE 2 MRM LM TR3_1-E |
| Subject ID: | SU002885 |
| Subject Type: | Amphibian |
| Subject Species: | Xenopus laevis |
| Taxonomy ID: | 8355 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002885 |
| Subject Type: | Amphibian |
| Subject Species: | Xenopus laevis |
| Taxonomy ID: | 8355 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 2021-02-01 JL14 SO WE 2 MRM LM TR3_1-E | SA297371 | FL036050 | RE | Sample type |
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 |
