#METABOLOMICS WORKBENCH cd_705_20240224_142316 DATATRACK_ID:4671 STUDY_ID:ST003120 ANALYSIS_ID:AN005114 PROJECT_ID:PR001938 VERSION 1 CREATED_ON February 27, 2024, 4:40 am #PROJECT PR:PROJECT_TITLE Role of central carbon metabolism in embryonic development PR:PROJECT_SUMMARY This work aims to understand how central carbon metabolism plays a crucial role PR:PROJECT_SUMMARY in germ layer fate specification and morphogenesis during gastrulation. In this PR:PROJECT_SUMMARY project, we manipulated central carbon metabolism using different glucose PR:PROJECT_SUMMARY concentrations and its inhibitors. To understand developmental phenotype of such PR:PROJECT_SUMMARY manipulations, we analysed the levels of intermediates of the glycolytic PR:PROJECT_SUMMARY pathway, oxidative phosphorylation, hexosamine biosynthetic pathway etc as well PR:PROJECT_SUMMARY as glucose epimers such as fucose, mannose, galactose. We later tested how PR:PROJECT_SUMMARY changes in these metabolite levels affected signalling pathways, important in PR:PROJECT_SUMMARY germ layer fate specification and subsequently their morphogenesis. PR:INSTITUTE Dept of Genetics, University of Cambridge PR:LAST_NAME Dingare PR:FIRST_NAME Chaitanya PR:ADDRESS Downing Site, Cambridge, Cambridgeshire, CB2 3EH, United Kingdom PR:EMAIL cd705@cam.ac.uk PR:PHONE +447916677460 #STUDY ST:STUDY_TITLE Mannose is crucial for mesoderm specification and symmetry breaking in ST:STUDY_TITLE gastruloids. ST:STUDY_SUMMARY Patterning and growth are fundamental features of embryonic development that ST:STUDY_SUMMARY must be tightly coordinated. To understand how metabolism impacts early mesoderm ST:STUDY_SUMMARY development, we used mouse embryonic stem cell-derived gastruloids, that ST:STUDY_SUMMARY co-expressed glucose transporters with the mesodermal marker T/Bra. While the ST:STUDY_SUMMARY glucose mimic, 2-deoxy-D-glucose (2-DG), blocked T/Bra expression and abolished ST:STUDY_SUMMARY axial elongation in gastruloids, removal of glucose did not phenocopy 2-DG ST:STUDY_SUMMARY treatment despite a decline in glycolytic intermediates occurring under both ST:STUDY_SUMMARY conditions. As 2-DG could also act as a competitive inhibitor of mannose in ST:STUDY_SUMMARY protein glycosylation, we added mannose together with 2-DG and found that it ST:STUDY_SUMMARY could rescue the mesoderm specification both in vivo and in vitro. We further ST:STUDY_SUMMARY showed that blocking production and intracellular recycling of mannose abrogated ST:STUDY_SUMMARY mesoderm specification. Proteomics analysis revealed that mannose reversed ST:STUDY_SUMMARY glycosylation of the Wnt pathway regulator, Secreted Frizzled Receptor, Frzb. ST:STUDY_SUMMARY Our study showed how mannose is crucial for mesoderm specification in ST:STUDY_SUMMARY gastruloids. ST:INSTITUTE Dept of Genetics, University of Cambridge ST:LAST_NAME Dingare ST:FIRST_NAME Chaitanya ST:ADDRESS Downing Site, Cambridge, Cambridgeshire, CB2 3EH, United Kingdom ST:EMAIL cd705@cam.ac.uk ST:PHONE +447916677460 ST:PUBLICATIONS https://doi.org/10.1101/2023.06.05.543730 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - CD041_001 Sample source:Mouse Embryonic Stem Cells | Treatment:No Glucose RAW_FILE_NAME(Raw_File_Name)=CD041_001.mzML SUBJECT_SAMPLE_FACTORS - CD041_002 Sample source:Mouse Embryonic Stem Cells | Treatment:1X Glucose RAW_FILE_NAME(Raw_File_Name)=CD041_002.mzML SUBJECT_SAMPLE_FACTORS - CD041_003 Sample source:Mouse Embryonic Stem Cells | Treatment:3X Glucose RAW_FILE_NAME(Raw_File_Name)=CD041_003.mzML SUBJECT_SAMPLE_FACTORS - CD041_004 Sample source:Mouse Embryonic Stem Cells | Treatment:2DG_5mM RAW_FILE_NAME(Raw_File_Name)=CD041_004.mzML SUBJECT_SAMPLE_FACTORS - CD042_001 Sample source:Mouse Embryonic Stem Cells | Treatment:No Glucose RAW_FILE_NAME(Raw_File_Name)=CD042_001.mzML SUBJECT_SAMPLE_FACTORS - CD042_002 Sample source:Mouse Embryonic Stem Cells | Treatment:1X Glucose RAW_FILE_NAME(Raw_File_Name)=CD042_002.mzML SUBJECT_SAMPLE_FACTORS - CD042_003 Sample source:Mouse Embryonic Stem Cells | Treatment:3X Glucose RAW_FILE_NAME(Raw_File_Name)=CD042_003.mzML SUBJECT_SAMPLE_FACTORS - CD042_004 Sample source:Mouse Embryonic Stem Cells | Treatment:2DG_5mM RAW_FILE_NAME(Raw_File_Name)=CD042_004.mzML SUBJECT_SAMPLE_FACTORS - CD043_001 Sample source:Mouse Embryonic Stem Cells | Treatment:No Glucose RAW_FILE_NAME(Raw_File_Name)=CD043_001.mzML SUBJECT_SAMPLE_FACTORS - CD043_002 Sample source:Mouse Embryonic Stem Cells | Treatment:1X Glucose RAW_FILE_NAME(Raw_File_Name)=CD043_002.mzML SUBJECT_SAMPLE_FACTORS - CD043_003 Sample source:Mouse Embryonic Stem Cells | Treatment:3X Glucose RAW_FILE_NAME(Raw_File_Name)=CD043_003.mzML SUBJECT_SAMPLE_FACTORS - CD043_004 Sample source:Mouse Embryonic Stem Cells | Treatment:2DG_5mM RAW_FILE_NAME(Raw_File_Name)=CD043_004.mzML SUBJECT_SAMPLE_FACTORS - CD044_001 Sample source:Mouse Embryonic Stem Cells | Treatment:No Glucose RAW_FILE_NAME(Raw_File_Name)=CD044_001.mzML SUBJECT_SAMPLE_FACTORS - CD044_002 Sample source:Mouse Embryonic Stem Cells | Treatment:1X Glucose RAW_FILE_NAME(Raw_File_Name)=CD044_002.mzML SUBJECT_SAMPLE_FACTORS - CD044_003 Sample source:Mouse Embryonic Stem Cells | Treatment:3X Glucose RAW_FILE_NAME(Raw_File_Name)=CD044_003.mzML SUBJECT_SAMPLE_FACTORS - CD044_004 Sample source:Mouse Embryonic Stem Cells | Treatment:2DG_5mM RAW_FILE_NAME(Raw_File_Name)=CD044_004.mzML SUBJECT_SAMPLE_FACTORS - QC08 Sample source:Mouse Embryonic Stem Cells | Treatment:Quality Control RAW_FILE_NAME(Raw_File_Name)=QC08.mzML SUBJECT_SAMPLE_FACTORS - QC09 Sample source:Mouse Embryonic Stem Cells | Treatment:Quality Control RAW_FILE_NAME(Raw_File_Name)=QC09.mzML SUBJECT_SAMPLE_FACTORS - QC10 Sample source:Mouse Embryonic Stem Cells | Treatment:Quality Control RAW_FILE_NAME(Raw_File_Name)=QC010.mzML SUBJECT_SAMPLE_FACTORS - QC11 Sample source:Mouse Embryonic Stem Cells | Treatment:Quality Control RAW_FILE_NAME(Raw_File_Name)=QC011.mzML SUBJECT_SAMPLE_FACTORS - QC12 Sample source:Mouse Embryonic Stem Cells | Treatment:Quality Control RAW_FILE_NAME(Raw_File_Name)=QC012.mzML SUBJECT_SAMPLE_FACTORS - Proc_Blank_start Sample source:Mouse Embryonic Stem Cells | Treatment:Blank RAW_FILE_NAME(Raw_File_Name)=Proc_Blank_start.mzML #COLLECTION CO:COLLECTION_SUMMARY Day 4 gastruloids from 2 plates were collected (approximately 45-48 gastruloids, CO:COLLECTION_SUMMARY per treatment, per biological replicate) (N, biological replicate = 4) into ice CO:COLLECTION_SUMMARY cold PBS in a clean glass petri dish, washed once and transferred to another CO:COLLECTION_SUMMARY glass petri dish on ice containing cold PBS. Gastruloids were collected, briefly CO:COLLECTION_SUMMARY centrifuged to settle them down and the PBS was completely removed. Gastruloids CO:COLLECTION_SUMMARY were snap-frozen in liquid nitrogen. To count the number of cells in each CO:COLLECTION_SUMMARY gastruloid, 5 gastruloids from each batch and each treatment, were collected, CO:COLLECTION_SUMMARY trypsinised for 1 min in 50 ul of 0.05% trypsin (25300054, ThermoFisher CO:COLLECTION_SUMMARY Scientific) and neutralised using 950 ul of warm N2B27. Cells were immediately CO:COLLECTION_SUMMARY counted using the Neubauer haemocytometer chamber. Snap-frozen gastruloids were CO:COLLECTION_SUMMARY sent to EMBL-Heidelberg, Germany, for the metabolomics analysis. Reagents: LC-MS CO:COLLECTION_SUMMARY grade water, acetonitrile and methanol were obtained from Th. Geyer (Germany). CO:COLLECTION_SUMMARY High-purity ammonium acetate, ammonium hydroxide, and formic acid were purchased CO:COLLECTION_SUMMARY from Merck (Germany). Stable isotope labelled amino acids (MSK-MET1-1; Cambridge CO:COLLECTION_SUMMARY Isotope Laboratories, MA, USA) were used as internal standards for untargeted CO:COLLECTION_SUMMARY metabolomics. Sample preparation: Metabolite extraction was performed by CO:COLLECTION_SUMMARY addition of 200 µL 80 % methanol (including 2 % (v/v) internal standards) and CO:COLLECTION_SUMMARY subsequent homogenization on dry ice via a bead beater (FastPrep-24; MP CO:COLLECTION_SUMMARY Biomedicals, CA, USA) at 6.0 m/s (5 x 30 s, 5 min pause time) using 1.0 mm CO:COLLECTION_SUMMARY zirconia/glass beads (Biospec Products, OK, USA). After centrifugation for 10 CO:COLLECTION_SUMMARY min at 15,000 ? g and 4 °C with a 5415R microcentrifuge (Eppendorf, Hamburg, CO:COLLECTION_SUMMARY Germany), supernatants were transferred and the remaining sample residues were CO:COLLECTION_SUMMARY reextracted with 200 µL acetonitrile:methanol:water (2:2:1, v/v) containing 1 % CO:COLLECTION_SUMMARY (v/v) formic acid using identical settings for homogenization and CO:COLLECTION_SUMMARY centrifugation. Corresponding supernatants of both extraction steps were CO:COLLECTION_SUMMARY combined and dried under a stream of nitrogen. Dried samples were reconstituted CO:COLLECTION_SUMMARY in 60 µL acetonitrile:methanol:water (2:2:1, v/v), vortexed for 5 min, CO:COLLECTION_SUMMARY centrifuged, and transferred to analytical glass vials. The LC-MS/MS analysis CO:COLLECTION_SUMMARY was initiated within one hour after the completion of the sample preparation. CO:SAMPLE_TYPE Embryonic cells CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY Mouse embryonic stem cell derived gastruloids were treated with different TR:TREATMENT_SUMMARY glucose concentrations (0, 21.25mM and 63.75mM) and 5mM of 2-deoxy-D-Glucose TR:TREATMENT_SUMMARY between day 3 and 4 of their developmental time window. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY LC-MS/MS analysis was performed on a Vanquish UHPLC system coupled to an SP:SAMPLEPREP_SUMMARY Orbitrap Exploris?240 high-resolution mass spectrometer (Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific, MA, USA) in negative ESI (electrospray ionization) mode. SP:SAMPLEPREP_SUMMARY Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC SP:SAMPLEPREP_SUMMARY column (Waters, MA, USA; 2.1?mm x 100 mm, 1.7 µm) at a flow rate of SP:SAMPLEPREP_SUMMARY 0.25?mL/min. The mobile phase consisted of water:acetonitrile (9:1, v/v;?mobile SP:SAMPLEPREP_SUMMARY phase phase A) and acetonitrile:water (9:1, v/v;?mobile phase?B), which were SP:SAMPLEPREP_SUMMARY modified with a total buffer concentration of 10 mM ammonium acetate. The SP:SAMPLEPREP_SUMMARY aqueous portion of each mobile phase was adjusted to pH 9.0 via addition of SP:SAMPLEPREP_SUMMARY ammonium hydroxide. The following gradient (20 min total run time including SP:SAMPLEPREP_SUMMARY re-equilibration) was applied (time?[min]/%B): 0/95, 2/95, 14.5/60, 16/60, SP:SAMPLEPREP_SUMMARY 16.5/95, 20/95. Column temperature was maintained at 40°C, the autosampler was SP:SAMPLEPREP_SUMMARY set to 4°C and sample injection volume was 7 µL. Analytes were recorded via a SP:SAMPLEPREP_SUMMARY full scan with a mass resolving power of 120,000 over a mass range from 60 – SP:SAMPLEPREP_SUMMARY 900 m/z (scan time: 100 ms, RF lens: 70%). To obtain MS/MS fragment spectra, SP:SAMPLEPREP_SUMMARY data-dependant acquisition was carried out (resolving power: 15,000; scan time: SP:SAMPLEPREP_SUMMARY 22 ms; stepped collision energies [%]: 30/50/70; cycle time: 900 ms). Ion source SP:SAMPLEPREP_SUMMARY parameters were set to the following values: spray voltage: 4100 V (positive SP:SAMPLEPREP_SUMMARY mode) / -3500?V (negative mode), sheath gas: 30 psi, auxiliary gas: 5 psi, sweep SP:SAMPLEPREP_SUMMARY gas: 0 psi, ion transfer tube temperature: 350°C, vaporizer temperature: SP:SAMPLEPREP_SUMMARY 300°C. All experimental samples were measured in a randomized manner. Pooled SP:SAMPLEPREP_SUMMARY quality control (QC) samples were prepared by mixing equal aliquots from each SP:SAMPLEPREP_SUMMARY processed sample. Multiple QCs were injected at the beginning of the analysis in SP:SAMPLEPREP_SUMMARY order to equilibrate the analytical system. A QC sample was analyzed after every SP:SAMPLEPREP_SUMMARY 5th experimental sample to monitor instrument performance?throughout the SP:SAMPLEPREP_SUMMARY sequence. For determination of background signals and subsequent background SP:SAMPLEPREP_SUMMARY subtraction, an additional processed blank sample was recorded. Data was SP:SAMPLEPREP_SUMMARY processed using MS-DIAL and raw peak intensities for relative metabolite SP:SAMPLEPREP_SUMMARY quantification 52. Feature identification was based on accurate mass, isotope SP:SAMPLEPREP_SUMMARY pattern, MS/MS fragment scoring and retention time matching to an inhouse SP:SAMPLEPREP_SUMMARY library. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Vanquish UHPLC CH:COLUMN_NAME Atlantis Premier BEH Z-HILIC column Waters, MA, USA; 2.1 mm x 100 mm, 1.7 µm CH:SOLVENT_A water:acetonitrile 9:1, v/v mobile phase CH:SOLVENT_B acetonitrile:water 9:1, v/v; mobile phase CH:FLOW_GRADIENT time [min]/%B - 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95 CH:FLOW_RATE 0.25mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fisher Scientific Orbitrap Exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Analytes were recorded via a full scan with a mass resolving power of 120,000 MS:MS_COMMENTS over a mass range from 60 – 900 m/z (scan time: 100 ms, RF lens: 70%). To MS:MS_COMMENTS obtain MS/MS fragment spectra, data-dependant acquisition was carried out MS:MS_COMMENTS (resolving power: 15,000; scan time: 22 ms; stepped collision energies [%]: MS:MS_COMMENTS 30/50/70; cycle time: 900 ms). Ion source parameters were set to the following MS:MS_COMMENTS values: spray voltage: 4100 V (positive mode) / -3500 V (negative mode), MS:MS_COMMENTS sheath gas: 30 psi, auxiliary gas: 5 psi, sweep gas: 0 psi, ion transfer tube MS:MS_COMMENTS temperature: 350°C, vaporizer temperature: 300°C. All experimental samples MS:MS_COMMENTS were measured in a randomized manner. Pooled quality control (QC) samples were MS:MS_COMMENTS prepared by mixing equal aliquots from each processed sample. Multiple QCs were MS:MS_COMMENTS injected at the beginning of the analysis in order to equilibrate the analytical MS:MS_COMMENTS system. A QC sample was analyzed after every 5th experimental sample to monitor MS:MS_COMMENTS instrument performance throughout the sequence. For determination of MS:MS_COMMENTS background signals and subsequent background subtraction, an additional MS:MS_COMMENTS processed blank sample was recorded. Data was processed using MS-DIAL and raw MS:MS_COMMENTS peak intensities for relative metabolite quantification. Feature identification MS:MS_COMMENTS was based on accurate mass, isotope pattern, MS/MS fragment scoring and MS:MS_COMMENTS retention time matching to an inhouse library. MS:MS_RESULTS_FILE ST003120_AN005114_Results.txt UNITS:counts per second (cps) Has m/z:Yes Has RT:Yes RT units:Minutes #END