Summary of Study ST003120
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 PR001938. The data can be accessed directly via it's Project DOI: 10.21228/M86T69 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.
| Study ID | ST003120 |
| Study Title | Mannose is crucial for mesoderm specification and symmetry breaking in gastruloids. |
| Study Summary | Patterning and growth are fundamental features of embryonic development that must be tightly coordinated. To understand how metabolism impacts early mesoderm development, we used mouse embryonic stem cell-derived gastruloids, that co-expressed glucose transporters with the mesodermal marker T/Bra. While the glucose mimic, 2-deoxy-D-glucose (2-DG), blocked T/Bra expression and abolished axial elongation in gastruloids, removal of glucose did not phenocopy 2-DG treatment despite a decline in glycolytic intermediates occurring under both conditions. As 2-DG could also act as a competitive inhibitor of mannose in protein glycosylation, we added mannose together with 2-DG and found that it could rescue the mesoderm specification both in vivo and in vitro. We further showed that blocking production and intracellular recycling of mannose abrogated mesoderm specification. Proteomics analysis revealed that mannose reversed glycosylation of the Wnt pathway regulator, Secreted Frizzled Receptor, Frzb. Our study showed how mannose is crucial for mesoderm specification in gastruloids. |
| Institute | Dept of Genetics, University of Cambridge |
| Last Name | Dingare |
| First Name | Chaitanya |
| Address | Downing Site, Cambridge, Cambridgeshire, CB2 3EH, United Kingdom |
| cd705@cam.ac.uk | |
| Phone | +447916677460 |
| Submit Date | 2024-02-24 |
| Publications | https://doi.org/10.1101/2023.06.05.543730 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-03-13 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001938 |
| Project DOI: | doi: 10.21228/M86T69 |
| Project Title: | Role of central carbon metabolism in embryonic development |
| Project Summary: | This work aims to understand how central carbon metabolism plays a crucial role in germ layer fate specification and morphogenesis during gastrulation. In this project, we manipulated central carbon metabolism using different glucose concentrations and its inhibitors. To understand developmental phenotype of such manipulations, we analysed the levels of intermediates of the glycolytic pathway, oxidative phosphorylation, hexosamine biosynthetic pathway etc as well as glucose epimers such as fucose, mannose, galactose. We later tested how changes in these metabolite levels affected signalling pathways, important in germ layer fate specification and subsequently their morphogenesis. |
| Institute: | Dept of Genetics, University of Cambridge |
| Last Name: | Dingare |
| First Name: | Chaitanya |
| Address: | Downing Site, Cambridge, Cambridgeshire, CB2 3EH, United Kingdom |
| Email: | cd705@cam.ac.uk |
| Phone: | +447916677460 |
Subject:
| Subject ID: | SU003237 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment |
|---|---|---|---|
| SA338087 | CD044_002 | Mouse Embryonic Stem Cells | 1X Glucose |
| SA338088 | CD043_002 | Mouse Embryonic Stem Cells | 1X Glucose |
| SA338089 | CD042_002 | Mouse Embryonic Stem Cells | 1X Glucose |
| SA338090 | CD041_002 | Mouse Embryonic Stem Cells | 1X Glucose |
| SA338091 | CD044_004 | Mouse Embryonic Stem Cells | 2DG_5mM |
| SA338092 | CD042_004 | Mouse Embryonic Stem Cells | 2DG_5mM |
| SA338093 | CD043_004 | Mouse Embryonic Stem Cells | 2DG_5mM |
| SA338094 | CD041_004 | Mouse Embryonic Stem Cells | 2DG_5mM |
| SA338095 | CD042_003 | Mouse Embryonic Stem Cells | 3X Glucose |
| SA338096 | CD044_003 | Mouse Embryonic Stem Cells | 3X Glucose |
| SA338097 | CD041_003 | Mouse Embryonic Stem Cells | 3X Glucose |
| SA338098 | CD043_003 | Mouse Embryonic Stem Cells | 3X Glucose |
| SA338099 | Proc_Blank_start | Mouse Embryonic Stem Cells | Blank |
| SA338100 | CD043_001 | Mouse Embryonic Stem Cells | No Glucose |
| SA338101 | CD042_001 | Mouse Embryonic Stem Cells | No Glucose |
| SA338102 | CD044_001 | Mouse Embryonic Stem Cells | No Glucose |
| SA338103 | CD041_001 | Mouse Embryonic Stem Cells | No Glucose |
| SA338104 | QC12 | Mouse Embryonic Stem Cells | Quality Control |
| SA338105 | QC10 | Mouse Embryonic Stem Cells | Quality Control |
| SA338106 | QC08 | Mouse Embryonic Stem Cells | Quality Control |
| SA338107 | QC09 | Mouse Embryonic Stem Cells | Quality Control |
| SA338108 | QC11 | Mouse Embryonic Stem Cells | Quality Control |
| Showing results 1 to 22 of 22 |
Collection:
| Collection ID: | CO003230 |
| Collection Summary: | Day 4 gastruloids from 2 plates were collected (approximately 45-48 gastruloids, per treatment, per biological replicate) (N, biological replicate = 4) into ice cold PBS in a clean glass petri dish, washed once and transferred to another glass petri dish on ice containing cold PBS. Gastruloids were collected, briefly centrifuged to settle them down and the PBS was completely removed. Gastruloids were snap-frozen in liquid nitrogen. To count the number of cells in each gastruloid, 5 gastruloids from each batch and each treatment, were collected, trypsinised for 1 min in 50 µl of 0.05% trypsin (25300054, ThermoFisher Scientific) and neutralised using 950 µl of warm N2B27. Cells were immediately counted using the Neubauer haemocytometer chamber. |
| Collection Protocol Filename: | Sample_Collection_CD.pdf |
| Sample Type: | Embryonic cells |
| Storage Conditions: | Described in summary |
Treatment:
| Treatment ID: | TR003246 |
| Treatment Summary: | Mouse embryonic stem cell derived gastruloids were treated with different glucose concentrations (0, 21.25mM and 63.75mM) and 5mM of 2-deoxy-D-Glucose between day 3 and 4 of their developmental time window. |
Sample Preparation:
| Sampleprep ID: | SP003244 |
| Sampleprep Summary: | Sample preparation: Metabolite extraction was performed by addition of 200 µL 80 % methanol (including 2 % (v/v) internal standards) and subsequent homogenization on dry ice via a bead beater (FastPrep-24; MP Biomedicals, CA, USA) at 6.0 m/s (5 x 30 s, 5 min pause time) using 1.0 mm zirconia/glass beads (Biospec Products, OK, USA). After centrifugation for 10 min at 15,000 g and 4 °C with a 5415R microcentrifuge (Eppendorf, Hamburg, Germany), supernatants were transferred and the remaining sample residues were reextracted with 200 µL acetonitrile:methanol:water (2:2:1, v/v) containing 1 % (v/v) formic acid using identical settings for homogenization and centrifugation. Corresponding supernatants of both extraction steps were combined and dried under a stream of nitrogen. Dried samples were reconstituted in 60 µL acetonitrile:methanol:water (2:2:1, v/v), vortexed for 5 min, centrifuged, and transferred to analytical glass vials. The LC-MS/MS analysis was initiated within one hour after the completion of the sample preparation. |
| Sampleprep Protocol Filename: | Sample_Collection_CD.pdf |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | Described in summary |
Combined analysis:
| Analysis ID | AN005114 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Vanquish UHPLC |
| Column | Atlantis Premier BEH Z-HILIC (100 x 2.1 mm, 1.7 um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Fisher Scientific Orbitrap Exploris 240 |
| Ion Mode | NEGATIVE |
| Units | counts per second (cps) |
Chromatography:
| Chromatography ID: | CH003870 |
| Chromatography Summary: | Snap-frozen gastruloids were sent to EMBL-Heidelberg, Germany, for the metabolomics analysis. Reagents: LC-MS grade water, acetonitrile and methanol were obtained from Th. Geyer (Germany). High-purity ammonium acetate, ammonium hydroxide, and formic acid were purchased from Merck (Germany). Stable isotope labelled amino acids (MSK-MET1-1; Cambridge Isotope Laboratories, MA, USA) were used as internal standards for untargeted metabolomics. LC-MS/MS analysis was performed on a Vanquish UHPLC system coupled to an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA) in negative ESI (electrospray ionization) mode. Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC column (Waters, MA, USA; 2.1?mm x 100 mm, 1.7 µm) at a flow rate of 0.25 mL/min. The mobile phase consisted of water:acetonitrile (9:1, v/v; mobile phase A) and acetonitrile:water (9:1, v/v; mobile phase B), which were modified with a total buffer concentration of 10 mM ammonium acetate. The aqueous portion of each mobile phase was adjusted to pH 9.0 via addition of ammonium hydroxide. The following gradient (20 min total run time including re-equilibration) was applied (time[min]/%B): 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95. Column temperature was maintained at 40°C, the autosampler was set to 4°C and sample injection volume was 7 µL. |
| Methods Filename: | LC_MS_MS_Full_Protocol.pdf |
| Instrument Name: | Vanquish UHPLC |
| Column Name: | Atlantis Premier BEH Z-HILIC (100 x 2.1 mm, 1.7 um) |
| Column Temperature: | 40 |
| Flow Gradient: | time [min]/%B - 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95 |
| Flow Rate: | 0.25mL/min |
| Solvent A: | water:acetonitrile 9:1, v/v; 10mM ammonium acetate |
| Solvent B: | acetonitrile:water 9:1, v/v; 10mM ammonium acetate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004851 |
| Analysis ID: | AN005114 |
| Instrument Name: | Thermo Fisher Scientific Orbitrap Exploris 240 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Analytes were recorded via a full scan with a mass resolving power of 120,000 over a mass range from 60 – 900 m/z (scan time: 100 ms, RF lens: 70%). To obtain MS/MS fragment spectra, data-dependant acquisition was carried out (resolving power: 15,000; scan time: 22 ms; stepped collision energies [%]: 30/50/70; cycle time: 900 ms). Ion source parameters were set to the following values: spray voltage: 4100 V (positive mode) / -3500 V (negative mode), sheath gas: 30 psi, auxiliary gas: 5 psi, sweep gas: 0 psi, ion transfer tube temperature: 350°C, vaporizer temperature: 300°C. All experimental samples were measured in a randomized manner. Pooled quality control (QC) samples were prepared by mixing equal aliquots from each processed sample. Multiple QCs were injected at the beginning of the analysis in order to equilibrate the analytical system. A QC sample was analyzed after every 5th experimental sample to monitor instrument performance throughout the sequence. For determination of background signals and subsequent background subtraction, an additional processed blank sample was recorded. Data was processed using MS-DIAL and raw peak intensities for relative metabolite quantification. Feature identification was based on accurate mass, isotope pattern, MS/MS fragment scoring and retention time matching to an inhouse library. (Data was acquired on both the positive and the negative modes and the respective raw files are deposited. However, we have processed the data acquired only on the negative mode.) |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | LC_MS_MS_Full_Protocol.pdf |
