Summary of Study ST003914

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 PR002449. The data can be accessed directly via it's Project DOI: 10.21228/M82Z52 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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST003914
Study TitleG1P promotes pentose phosphate pathway in CD8+ memory T cells via glycogen-G6PD phase separation and compartmentalization
Study SummaryGlucose-6-phosphate (G6P) is a key metabolic molecule that regulates reactive oxygen species (ROS) homeostasis by initiating the pentose phosphate pathway (PPP) to generate NADPH that converts H2O2 to water by providing hydrogen. While both glucose phosphorylation and glycogenolysis result in G6P production, here we show that G6P derived from glycogenolysis, rather than glucose phosphorylation, flows to PPP for ROS clearance in CD8+ memory T (Tm) cells and inflammatory macrophages. Mechanistically, glycogenolysis-produced G1P allosterically induces G6P dehydrogenase (G6PD) binding to glycogen, which together undergo liquid-liquid phase separation (LLPS) and recruit PPP enzymes, resulting in a compartmentalized reaction cascade. Based on mechanistic elucidation, we demonstrated that G1P can act as an antitumor immunotherapeutic agent by modulating memory fitness and maintenance of tumor-reactive CD8+ T cells in mice. These findings revealed an unusual function of glycogen metabolism, which is of paramount importance in the regulation of PPP and redox homeostasis in cells.
Institute
Peking Union Medical College
Last NameZhang
First NameChaoying
AddressNo. 5, Dongdan santiao, Dongcheng District, Beijing, Beijing, Beijing, 100005, China
Emailzhouyaboqq@gmail.com
Phone860169156464
Submit Date2025-05-08
Raw Data AvailableYes
Raw Data File Type(s)mzML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2025-05-26
Release Version1
Chaoying Zhang Chaoying Zhang
https://dx.doi.org/10.21228/M82Z52
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002449
Project DOI:doi: 10.21228/M82Z52
Project Title:Metabolomic profiling of glycogenolysis-derived G1P promotes pentose phosphate pathway in CD8+ memory T cell
Project Summary:Glucose-6-phosphate (G6P) is a key metabolic molecule that regulates reactive oxygen species (ROS) homeostasis by initiating the pentose phosphate pathway (PPP) to generate NADPH that converts H2O2 to water by providing hydrogen. While both glucose phosphorylation and glycogenolysis result in G6P production, here we show that G6P derived from glycogenolysis, rather than glucose phosphorylation, flows to PPP for ROS clearance in CD8+ memory T (Tm) cells and inflammatory macrophages. Mechanistically, glycogenolysis-produced G1P allosterically induces G6P dehydrogenase (G6PD) binding to glycogen, which together undergo liquid-liquid phase separation (LLPS) and recruit PPP enzymes, resulting in a compartmentalized reaction cascade. Based on mechanistic elucidation, we demonstrated that G1P can act as an antitumor immunotherapeutic agent by modulating memory fitness and maintenance of tumor-reactive CD8+ T cells in mice. These findings revealed an unusual function of glycogen metabolism, which is of paramount importance in the regulation of PPP and redox homeostasis in cells.
Institute:Peking Union Medical College
Last Name:Zhang
First Name:Chaoying
Address:No. 5, Dongdan santiao, Dongcheng District, Beijing, Beijing, Beijing, 100005, China
Email:zhouyaboqq@gmail.com
Phone:860169156464

Subject:

Subject ID:SU004049
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

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

mb_sample_id local_sample_id Experiment title Genotype Treatment condition Cell type
SA430583FigS1A_1Experiment 10 Wild Type Cellular glycogen isolation CD8 T cell
SA430584FigS1A_3Experiment 10 Wild Type Cellular glycogen isolation CD8 T cell
SA430585FigS1A_2Experiment 10 Wild Type Cellular glycogen isolation CD8 T cell
SA430592Fig_S1c_vec_2h3Experiment 11 Wild Type control treatment and treat 13C glucose for 2h CD8 T cell
SA430593Fig_S1c_vec_2h1Experiment 11 Wild Type control treatment and treat 13C glucose for 2h CD8 T cell
SA430594Fig_S1c_vec_2h2Experiment 11 Wild Type control treatment and treat 13C glucose for 2h CD8 T cell
SA430595Fig_S1c_vec_4h1Experiment 11 Wild Type control treatment and treat 13C glucose for 4h CD8 T cell
SA430596Fig_S1c_vec_4h2Experiment 11 Wild Type control treatment and treat 13C glucose for 4h CD8 T cell
SA430597Fig_S1c_vec_4h3Experiment 11 Wild Type control treatment and treat 13C glucose for 4h CD8 T cell
SA430586Fig_S1c_GPI_2h1Experiment 11 Wild Type GPI treatment and treat 13C glucose for 2h CD8 T cell
SA430587Fig_S1c_GPI_2h2Experiment 11 Wild Type GPI treatment and treat 13C glucose for 2h CD8 T cell
SA430588Fig_S1c_GPI_2h3Experiment 11 Wild Type GPI treatment and treat 13C glucose for 2h CD8 T cell
SA430589Fig_S1c_GPI_4h3Experiment 11 Wild Type GPI treatment and treat 13C glucose for 4h CD8 T cell
SA430590Fig_S1c_GPI_4h1Experiment 11 Wild Type GPI treatment and treat 13C glucose for 4h CD8 T cell
SA430591Fig_S1c_GPI_4h2Experiment 11 Wild Type GPI treatment and treat 13C glucose for 4h CD8 T cell
SA430604Fig_S1E_GYSKO1_2h3Experiment 12 sgGys1 GYS1 sgRNA and and treat 13C glucose for 2h CD8 T cell
SA430605Fig_S1E_GYSKO1_2h2Experiment 12 sgGys1 GYS1 sgRNA and and treat 13C glucose for 2h CD8 T cell
SA430606Fig_S1E_GYSKO1_2h1Experiment 12 sgGys1 GYS1 sgRNA and and treat 13C glucose for 2h CD8 T cell
SA430607Fig_S1E_SGgys1_4h2Experiment 12 sgGys1 GYS1 sgRNA and and treat 13C glucose for 4h CD8 T cell
SA430608Fig_S1E_SGgys1_4h3Experiment 12 sgGys1 GYS1 sgRNA and and treat 13C glucose for 4h CD8 T cell
SA430609Fig_S1E_SGgys1_4h1Experiment 12 sgGys1 GYS1 sgRNA and and treat 13C glucose for 4h CD8 T cell
SA430598Fig_S1E_SGCON_2h2Experiment 12 Wild Type control sgRNA and and treat 13C glucose for 2h CD8 T cell
SA430599Fig_S1E_SGCON_2h1Experiment 12 Wild Type control sgRNA and and treat 13C glucose for 2h CD8 T cell
SA430600Fig_S1E_SGCON_2h3Experiment 12 Wild Type control sgRNA and and treat 13C glucose for 2h CD8 T cell
SA430601Fig_S1E_SGCON_4h1Experiment 12 Wild Type control sgRNA and and treat 13C glucose for 4h CD8 T cell
SA430602Fig_S1E_SGCON_4h2Experiment 12 Wild Type control sgRNA and and treat 13C glucose for 4h CD8 T cell
SA430603Fig_S1E_SGCON_4h3Experiment 12 Wild Type control sgRNA and and treat 13C glucose for 4h CD8 T cell
SA430616Fig_S1H_SGGYS1_1Experiment 13 sgGys1 SG gys1 sgRNA BMDM
SA430617Fig_S1H_SGGYS1_2Experiment 13 sgGys1 SG gys1 sgRNA BMDM
SA430618Fig_S1H_SGGYS1_3Experiment 13 sgGys1 SG gys1 sgRNA BMDM
SA430619Fig_S1G_SGGYS1_1Experiment 13 sgGys1 SG gys1 sgRNA CD8 T cell
SA430620Fig_S1G_SGGYS1_2Experiment 13 sgGys1 SG gys1 sgRNA CD8 T cell
SA430621Fig_S1G_SGGYS1_3Experiment 13 sgGys1 SG gys1 sgRNA CD8 T cell
SA430610Fig_S1H_SGCTRL_1Experiment 13 Wild Type control sgRNA BMDM
SA430611Fig_S1H_SGCTRL_3Experiment 13 Wild Type control sgRNA BMDM
SA430612Fig_S1H_SGCTRL_2Experiment 13 Wild Type control sgRNA BMDM
SA430613Fig_S1G_SGCTRL_1Experiment 13 Wild Type control sgRNA CD8 T cell
SA430614Fig_S1G_SGCTRL_2Experiment 13 Wild Type control sgRNA CD8 T cell
SA430615Fig_S1G_SGCTRL_3Experiment 13 Wild Type control sgRNA CD8 T cell
SA430628Fig_S1i_dmso_3Experiment 14 Wild Type control treatment CD8 T cell
SA430629Fig_S1i_dmso_2Experiment 14 Wild Type control treatment CD8 T cell
SA430630Fig_S1i_dmso_1Experiment 14 Wild Type control treatment CD8 T cell
SA430622Fig_S1i_Gpi_g1p_2Experiment 14 Wild Type GPI and G1P treatment CD8 T cell
SA430623Fig_S1i_Gpi_g1p_1Experiment 14 Wild Type GPI and G1P treatment CD8 T cell
SA430624Fig_S1i_Gpi_g1p_3Experiment 14 Wild Type GPI and G1P treatment CD8 T cell
SA430625Fig_S1i_Gpi_2Experiment 14 Wild Type GPI treatment CD8 T cell
SA430626Fig_S1i_Gpi_3Experiment 14 Wild Type GPI treatment CD8 T cell
SA430627Fig_S1i_Gpi_1Experiment 14 Wild Type GPI treatment CD8 T cell
SA430637Fig_S1L_PGM1SG_G6P_2Experiment 15 sgPGM1 PGM1 sgRNA and G6P LNP treatment BMDM
SA430638Fig_S1L_PGM1SG_G6P_1Experiment 15 sgPGM1 PGM1 sgRNA and G6P LNP treatment BMDM
SA430639Fig_S1L_PGM1SG_G6P_3Experiment 15 sgPGM1 PGM1 sgRNA and G6P LNP treatment BMDM
SA430640Fig_S1K_SGPGM1_G6P_3Experiment 15 sgPGM1 PGM1 sgRNA and G6P LNP treatment CD8 T cell
SA430641Fig_S1K_SGPGM1_G6P_1Experiment 15 sgPGM1 PGM1 sgRNA and G6P LNP treatment CD8 T cell
SA430642Fig_S1K_SGPGM1_G6P_2Experiment 15 sgPGM1 PGM1 sgRNA and G6P LNP treatment CD8 T cell
SA430643Fig_S1L_PGM1SG_3Experiment 15 sgPGM1 PGM1 sgRNA BMDM
SA430644Fig_S1L_PGM1SG_2Experiment 15 sgPGM1 PGM1 sgRNA BMDM
SA430645Fig_S1L_PGM1SG_1Experiment 15 sgPGM1 PGM1 sgRNA BMDM
SA430646Fig_S1K_SGPGM1_1Experiment 15 sgPGM1 PGM1 sgRNA CD8 T cell
SA430647Fig_S1K_SGPGM1_2Experiment 15 sgPGM1 PGM1 sgRNA CD8 T cell
SA430648Fig_S1K_SGPGM1_3Experiment 15 sgPGM1 PGM1 sgRNA CD8 T cell
SA430631Fig_S1L_SGNC_3Experiment 15 Wild Type control sgRNA BMDM
SA430632Fig_S1L_SGNC_2Experiment 15 Wild Type control sgRNA BMDM
SA430633Fig_S1L_SGNC_1Experiment 15 Wild Type control sgRNA BMDM
SA430634Fig_S1K_SGNC_1Experiment 15 Wild Type control sgRNA CD8 T cell
SA430635Fig_S1K_SGNC_3Experiment 15 Wild Type control sgRNA CD8 T cell
SA430636Fig_S1K_SGNC_2Experiment 15 Wild Type control sgRNA CD8 T cell
SA430652Fig1_S1O_GPI_1Experiment 16 Wild Type control treatment CD8 T cell
SA430653Fig1_S1O_GPI_2Experiment 16 Wild Type control treatment CD8 T cell
SA430654Fig1_S1O_GPI_3Experiment 16 Wild Type control treatment CD8 T cell
SA430649Fig1_S1O_VEC_3Experiment 16 Wild Type GPI treatment CD8 T cell
SA430650Fig1_S1O_VEC_1Experiment 16 Wild Type GPI treatment CD8 T cell
SA430651Fig1_S1O_VEC_2Experiment 16 Wild Type GPI treatment CD8 T cell
SA430658Fig1_S1S_BLANK NP_1Experiment 17 Wild Type control NP CD8 T cell
SA430659Fig1_S1S_BLANK NP_2Experiment 17 Wild Type control NP CD8 T cell
SA430660Fig1_S1S_BLANK NP_3Experiment 17 Wild Type control NP CD8 T cell
SA430655Fig1_S1S_G1P NP_1Experiment 17 Wild Type G1P LNP treatment CD8 T cell
SA430656Fig1_S1S_G1P NP_3Experiment 17 Wild Type G1P LNP treatment CD8 T cell
SA430657Fig1_S1S_G1P NP_2Experiment 17 Wild Type G1P LNP treatment CD8 T cell
SA430661Fig_S4i_D421A_2Experiment 18 D421A G6PD D421 KI CD8 T cell
SA430662Fig_S4i_D421A_3Experiment 18 D421A G6PD D421 KI CD8 T cell
SA430663Fig_S4i_D421A_1Experiment 18 D421A G6PD D421 KI CD8 T cell
SA430664Fig_S4i_WT_1Experiment 18 Wild Type G6PD WT KI CD8 T cell
SA430665Fig_S4i_WT_2Experiment 18 Wild Type G6PD WT KI CD8 T cell
SA430666Fig_S4i_WT_3Experiment 18 Wild Type G6PD WT KI CD8 T cell
SA430670Fig_S4m_DMSO_3Experiment 19 Wild Type control treatment CD8 T cell
SA430671Fig_S4m_DMSO_1Experiment 19 Wild Type control treatment CD8 T cell
SA430672Fig_S4m_DMSO_2Experiment 19 Wild Type control treatment CD8 T cell
SA430667Fig_S4m_GPI_1Experiment 19 Wild Type GPI treatment CD8 T cell
SA430668Fig_S4m_GPI_2Experiment 19 Wild Type GPI treatment CD8 T cell
SA430669Fig_S4m_GPI_3Experiment 19 Wild Type GPI treatment CD8 T cell
SA430673Fig_1b_2DG_1Experiment 1 Wild Type 2-deoxy-D-glucose (2-DG, 1 mM) CD8 T cell
SA430674Fig_1b_2DG_2Experiment 1 Wild Type 2-deoxy-D-glucose (2-DG, 1 mM) CD8 T cell
SA430675Fig_1b_2DG_3Experiment 1 Wild Type 2-deoxy-D-glucose (2-DG, 1 mM) CD8 T cell
SA430679Fig_1A_VEC_1Experiment 1 Wild Type control treatment CD8 T cell
SA430680Fig_1A_VEC_2Experiment 1 Wild Type control treatment CD8 T cell
SA430681Fig_1A_VEC_3Experiment 1 Wild Type control treatment CD8 T cell
SA430682Fig_1b_vec_1Experiment 1 Wild Type control treatment CD8 T cell
SA430683Fig_1b_vec_2Experiment 1 Wild Type control treatment CD8 T cell
SA430684Fig_1b_vec_3Experiment 1 Wild Type control treatment CD8 T cell
SA430676Fig_1A_HKi_1Experiment 1 Wild Type HK inhibitor Lonidamine (50 μM) CD8 T cell
Showing page 1 of 2     Results:    1  2  Next     Showing results 1 to 100 of 182

Collection:

Collection ID:CO004042
Collection Summary:Murine CD8⁺ T cells were isolated from the spleens of C57BL/6J mice using negative selection with magnetic-activated cell sorting (MACS; Mouse CD8⁺ T Cell Isolation Kit, Miltenyi Biotec, Germany). For the generation of stable gene knockouts in CD8⁺ T cells or interferon-gamma (IFN-γ)-stimulated immortalized bone marrow-derived macrophages (iBMDMs), single-guide RNAs (sgRNAs) were synthesized using the HiScribe® Quick T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2050, USA). A total of 1 × 10⁶ T cells or IFN-γ-stimulated iBMDMs were resuspended in 20 µL of P3 Primary Cell Nucleofector Solution (Lonza, for the 4D-Nucleofector system), and co-electroporated with 0.5 µg of sgRNA and recombinant Cas9 protein. Isolated CD8⁺ T cells were activated using anti-CD3/CD28 magnetic beads (Thermo Fisher Scientific, #11453) according to the manufacturer’s instructions. Briefly, 1 × 10⁶ purified splenic OT-I CD8⁺ T cells were cultured in 1 mL RPMI 1640 medium containing 25 µL of pre-washed anti-CD3/CD28 beads and 10 ng/mL interleukin-2 (IL-2; PeproTech, #212-12) for three days. Following activation, cells were harvested, washed three times with RPMI 1640 medium, and further cultured at 2 × 10⁵ cells/mL in the presence of 10 ng/mL interleukin-15 (IL-15; PeproTech, #210-15) in 12-well culture plates (Corning, #3336) for an additional four days to induce memory CD8⁺ T cell (T_m) differentiation. Fresh medium supplemented with cytokines was replaced daily during the induction process. Mouse iBMDMs were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS). For gene knockout experiments, 1 × 10⁶ IFN-γ-stimulated iBMDMs were subjected to nucleofection as described above, using sgRNAs and Cas9 protein.
Sample Type:Immune cell
Storage Conditions:On ice

Treatment:

Treatment ID:TR004058
Treatment Summary:Experiment 1: CD8⁺ memory T (T_m) cells were treated with hexokinase (HK) inhibitor lonidamine (50 μM) or 2-deoxy-D-glucose (2-DG, 1 mM) for 24 hours. Ribose-5-phosphate (R5P) and sedoheptulose-7-phosphate (S7P) were detected. Experiment 2: CD8⁺ T_m cells were cultured in uniformly labeled 13C₆-glucose for 7 days and then switched to unlabeled 12C-glucose for 2 or 4 hours. m+5 labeled R5P was detected. Experiment 3: Control (sgCtrl) or glycogen phosphorylase brain isoform (sgPygb) CD8⁺ T_m cells cultured in 13C₆-glucose were switched to 12C-glucose for 2 or 4 hours. m+5 labeled R5P was detected. Experiment 4: sgCtrl or sgPygb CD8⁺ T_m cells were treated with glucose-1-phosphate-loaded nanoparticles (G1P-NPs, 5 μM) for 24 hours. R5P and S7P levels were detected. Experiment 5: CD8⁺ T_m cells pretreated with G1P-NPs (5 μM) for 24 hours were cultured in 13C₆-glucose for 4 hours. m+5 R5P was detected. Experiment 6: CD8⁺ T_m cells were treated with or without 5% 1,6-hexanediol (1,6-HD) for 10 minutes and then cultured in 13C₆-glucose for 4 hours. m+5 R5P was detected. Experiment 7: Glycogen was isolated and dissolved from CD8⁺ T_m cells. R5P from cytosol and glycogen fractions was measured. Experiment 8: CD8⁺ T_m cells pretreated with glucose-6-phosphate isomerase inhibitor (GPI, 50 μM) for 12 hours were electroporated with 13C₆-glucose-6-phosphate (13C₆-G6P) and cultured for 4 hours. m+5 R5P was analyzed. Experiment 9: C57BL/6 mice were subcutaneously inoculated with 1×10⁶ Lewis lung carcinoma expressing ovalbumin (LLC-OVA) cells. Eight days later, mice were transferred with or without OT-I T cells and injected intraperitoneally with or without CD8α-targeted G1P-NPs (50 μg/kg) every two days. Mice were fasted overnight and then infused with 13C₆-glucose (12.5 mg/kg/min) via the tail vein for 4 hours. m+5 R5P was detected in ovalbumin (OVA)-specific CD8⁺ T_m cells from lymph nodes (LNs) and tumor-infiltrating macrophages. Experiment 10: CD8⁺ T_m cells were induced by interleukin-15 (IL-15) and cultured in 13C₆-glucose for 7 days, followed by hydrochloric acid treatment. The released m+6 labeled glucose was measured. Experiment 11: CD8⁺ T_m cells cultured in 13C₆-glucose were pretreated with GPI (50 μM) for 12 hours and then switched to 12C-glucose for 2 or 4 hours. m+5 labeled R5P was detected. Experiment 12: sgCtrl or glycogen synthase 1 (sgGys1) CD8⁺ T_m cells cultured in 13C₆-glucose were switched to 12C-glucose for 2 or 4 hours. m+5 labeled R5P was detected. Experiment 13: sgCtrl or sgGys1 CD8⁺ T_m cells or immortalized bone marrow-derived macrophages (iBMDM) were cultured in 13C₆-glucose for 4 hours. m+5 labeled R5P was detected. Experiment 14: CD8⁺ T_m cells pretreated with GPI (50 μM) for 12 hours were treated with G1P-NPs (5 μM) for 24 hours. R5P and S7P levels were measured. Experiment 15: sgCtrl or phosphoglucomutase 1 (sgPgm1) CD8⁺ T_m cells or iBMDMs were treated with glucose-6-phosphate-loaded nanoparticles (G6P-NPs, 5 μM) for 24 hours. R5P and S7P levels were measured. Experiment 16: CD8⁺ T_m cells cultured in 13C₆-glucose for 7 days were electroporated with a pcDNA-mouse-glucose-6-phosphate dehydrogenase-Flag (pcDNA-m-G6PD-Flag) plasmid. Cells were then treated with or without GPI (50 μM) for 12 hours and switched to 12C-glucose medium for 2 hours. After washing with phosphate-buffered saline (PBS), cells were crosslinked by ultraviolet C (UVC, 254 nm) at 0.3 J/cm² and lysed. Immunoprecipitation was performed using anti-Flag magnetic beads. m+6 and m+0 G1P bound to beads were detected. Experiment 17: iBMDMs pretreated with G1P-NPs (5 μM) for 24 hours were cultured in 13C₆-glucose for 4 hours. m+5 labeled R5P was detected. Experiment 18: Wild-type G6PD (G6PDWT) or knock-in G6PDD421A CD8⁺ T_m cells were cultured in 13C₆-glucose for 4 hours. m+5 labeled R5P was detected. Experiment 19: CD8⁺ T_m cells pretreated with GPI (50 μM) for 12 hours were cultured in 13C₆-glucose for 2 hours. m+6 labeled uridine diphosphate glucose (UDPG) was detected. Experiment 20: iBMDMs pretreated with GPI (50 μM) for 12 hours were electroporated with 13C₆-G6P for 4 hours. m+5 R5P was analyzed. Experiment 21: CD8⁺ T_m cells or iBMDMs pretreated with 5% 1,6-HD for 10 minutes were electroporated with 13C₆-G6P for 4 hours. m+5 R5P was analyzed.
Treatment Protocol Filename:BoHuang_Lab_C13_Extraction_and_Analysis_Protocol.pdf

Sample Preparation:

Sampleprep ID:SP004055
Sampleprep Summary:For metabolite analysis, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and extracted with ice-cold 80% methanol. The cell suspension was subjected to three freeze-thaw cycles using liquid nitrogen to ensure complete lysis. After centrifugation at 12,000 × g for 10 minutes at 4 °C, the supernatant containing metabolites was transferred to a new tube and stored at −80 °C until further analysis.
Sampleprep Protocol Filename:BoHuang_Lab_C13_Extraction_and_Analysis_Protocol.pdf
Processing Storage Conditions:On ice
Extraction Method:Cells were washed twice with ice-cold PBS and metabolites were extracted with ice-cold 80% methanol.
Extract Storage:-80℃

Chromatography:

Chromatography ID:CH004875
Chromatography Summary:Solvent A: 20 mM ammonium acetate and 15 mM ammonium hydroxide in water with 3% acetonitrile, pH 9.0; Solvent B: acetonitrile
Methods Filename:BoHuang_Lab_C13_Extraction_and_Analysis_Protocol.pdf
Instrument Name:Thermo Vanquish
Column Name:Waters Xbridge Amide (100 × 2.1mm, 3.5um)
Column Temperature:35
Flow Gradient:0 min, 85% B; 1.5 min, 85% B, 5.5 min, 30% B; 8 min, 30% B, 10 min, 85% B, and 12 min, 85% B
Flow Rate:0.2 mL/min
Solvent A:100% water/3% acetonitrile; 20 mM ammonium acetate; 15 mM ammonium hydroxide
Solvent B:100% acetonitrile
Chromatography Type:HILIC

Analysis:

Analysis ID:AN006427
Analysis Type:MS
Chromatography ID:CH004875
Num Factors:60
Num Metabolites:13
Units:Peak area
  logo