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.

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Study IDST003120
Study TitleMannose is crucial for mesoderm specification and symmetry breaking in gastruloids.
Study SummaryPatterning 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 NameDingare
First NameChaitanya
AddressDowning Site, Cambridge, Cambridgeshire, CB2 3EH, United Kingdom
Emailcd705@cam.ac.uk
Phone+447916677460
Submit Date2024-02-24
Publicationshttps://doi.org/10.1101/2023.06.05.543730
Raw Data AvailableYes
Raw Data File Type(s)mzML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-03-13
Release Version1
Chaitanya Dingare Chaitanya Dingare
https://dx.doi.org/10.21228/M86T69
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment
SA338087CD044_002Mouse Embryonic Stem Cells 1X Glucose
SA338088CD043_002Mouse Embryonic Stem Cells 1X Glucose
SA338089CD042_002Mouse Embryonic Stem Cells 1X Glucose
SA338090CD041_002Mouse Embryonic Stem Cells 1X Glucose
SA338091CD044_004Mouse Embryonic Stem Cells 2DG_5mM
SA338092CD042_004Mouse Embryonic Stem Cells 2DG_5mM
SA338093CD043_004Mouse Embryonic Stem Cells 2DG_5mM
SA338094CD041_004Mouse Embryonic Stem Cells 2DG_5mM
SA338095CD042_003Mouse Embryonic Stem Cells 3X Glucose
SA338096CD044_003Mouse Embryonic Stem Cells 3X Glucose
SA338097CD041_003Mouse Embryonic Stem Cells 3X Glucose
SA338098CD043_003Mouse Embryonic Stem Cells 3X Glucose
SA338099Proc_Blank_startMouse Embryonic Stem Cells Blank
SA338100CD043_001Mouse Embryonic Stem Cells No Glucose
SA338101CD042_001Mouse Embryonic Stem Cells No Glucose
SA338102CD044_001Mouse Embryonic Stem Cells No Glucose
SA338103CD041_001Mouse Embryonic Stem Cells No Glucose
SA338104QC12Mouse Embryonic Stem Cells Quality Control
SA338105QC10Mouse Embryonic Stem Cells Quality Control
SA338106QC08Mouse Embryonic Stem Cells Quality Control
SA338107QC09Mouse Embryonic Stem Cells Quality Control
SA338108QC11Mouse 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
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