Summary of Study ST001952
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 PR001239. The data can be accessed directly via it's Project DOI: 10.21228/M8JT51 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001952 |
| Study Title | GLS2KO vs WT mouse hepatocytes |
| Study Type | Genotype |
| Study Summary | Test the effect of GLS2 knockout in primary mouse hepatocytes. We isolated hepatocytes from GLS2 knockout and wild-type mice, and briefly applied media lacking L-glutamine (2 hours). Sixty minutes after resupplying Gln, metabolites were extracted and analyzed with the Mixed Mode method. Data were processed through XCMS, and features were filtered for p<0.01, fold change >2, and a minimum intensity of 1x10^6. Sorting by smallest p value, the first extracted ion chromatogram (EIC) with good chromatographic peak shape corresponded to 188.0567m/z at 24.41 min. Six putative IDs were within 3ppm of the experimentally observed m/z, representing two chemical formulas, none of which had documented retention times in training or test sets. Amongst these potential IDs, the Message Passing Neural Network (MPNN) model correctly predicted N-acetyl-L-glutamic acid as the most likely candidate, as verified by injection of purchased standards. The next most significant difference between GLS2KO vs WT was 117.0196m/z observed at 20.07 minutes. The model had been trained on 2/6 of the putative IDs. Despite the four additional isomers suggested, the model correctly selected succinate as reduced by GLS2 KO (Figure 5B). The third most significant hit corresponds to 171.0068m/z at 23.11 min. Although glycerol 1-P and 2-P are both potential hits, almost indistinguishable by the model, the large gap in retention times between these top hits and the Cl- adducts of threonate (+ isomers) is apparent, further supporting the correct identification as glycerol mono-phosphate. Expansion of the list to include p<0.05 leads to the identification of Glutamine, Glutamate, and other downstream metabolites known to be altered by GLS2 KO. |
| Institute | Pfizer |
| Last Name | Clasquin |
| First Name | Michelle |
| Address | 1 Portland St., Cambridge, MA 02139 |
| michelle.clasquin@pfizer.com | |
| Phone | 6174487289 |
| Submit Date | 2021-10-22 |
| Num Groups | 2 |
| Total Subjects | 6 |
| Num Males | 6 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-11-08 |
| Release Version | 1 |
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Project:
| Project ID: | PR001239 |
| Project DOI: | doi: 10.21228/M8JT51 |
| Project Title: | GLS2KO vs WT mouse hepatocytes |
| Project Type: | Genotype |
| Project Summary: | Test the effect of knocking out GLS2 in primary mouse heptocytes. This data set was generated by applying Mixed Mode chromatography coupled to a Q Exactive Plus Orbitrap Mass Spectrometer with enhanced MS resolution up to 280,000. The most significantly altered metabolites as identified by XCMS were used to test the ability of a Message Passing Neural Network (MPNN) Model to rank order metabolite IDs. |
| Institute: | Pfizer |
| Department: | Internal Medicine Research Unit, WRDM |
| Last Name: | Clasquin |
| First Name: | Michelle |
| Address: | 1 Portland St., Cambridge, MA 02139 |
| Email: | michelle.clasquin@pfizer.com |
| Phone: | 6174487289 |
| Contributors: | Gang Xing, Yuji Shi |
Subject:
| Subject ID: | SU002030 |
| 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 | Genotype |
|---|---|---|
| SA184050 | 6 | GLS2 KO |
| SA184051 | 5 | GLS2 KO |
| SA184052 | 4 | GLS2 KO |
| SA184053 | 2 | Wild-type |
| SA184054 | 3 | Wild-type |
| SA184055 | 1 | Wild-type |
| Showing results 1 to 6 of 6 |
Collection:
| Collection ID: | CO002023 |
| Collection Summary: | All samples extracted from mice were collected in accordance with regulations and established guidelines for humane treatment of research animals and were reviewed and approved by an Institutional Animal Care and Use Committee, Project Code HD-EX00066. Primary hepatocytes were isolated from male mice between 12-18 weeks of age by the two-step collagenase perfusion method. Mice were fasted 16 hours before the ex-periments. After isolation, cells were plated in M199 media with 10% FBS for 4 hours in 6-well plates pre-coated with collagen I. After cells attached to the plates, they were washed with glucose output media (GOM) (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 20 mM NaCO3, 25 mM HEPES pH 7.4, and 0.025% BSA), and incubated in fresh GOM for 2 hour. GOM media was replaced with fresh, pre-warmed GOM media and cellular treatments initiated. Hepatocytes were treated with 5mM unlabeled glutamine for 60 min. Hepatocytes were washed with ice cold PBS twice and immediately frozen in liquid nitrogen. |
| Sample Type: | Liver |
Treatment:
| Treatment ID: | TR002042 |
| Treatment Summary: | All samples extracted from mice were collected in accordance with regulations and established guidelines for humane treatment of research animals and were reviewed and approved by an Institutional Animal Care and Use Committee, Project Code HD-EX00066. Primary hepatocytes were isolated from male mice between 12-18 weeks of age by the two-step collagenase perfusion method. Mice were fasted 16 hours before the ex-periments. After isolation, cells were plated in M199 media with 10% FBS for 4 hours in 6-well plates pre-coated with collagen I. After cells attached to the plates, they were washed with glucose output media (GOM) (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 20 mM NaCO3, 25 mM HEPES pH 7.4, and 0.025% BSA), and incubated in fresh GOM for 2 hour. GOM media was replaced with fresh, pre-warmed GOM media and cellular treatments initiated. Hepatocytes were treated with 5mM unlabeled glutamine for 60 min. Hepatocytes were washed with ice cold PBS twice and immediately frozen in liquid nitrogen. |
Sample Preparation:
| Sampleprep ID: | SP002036 |
| Sampleprep Summary: | Metabolites were extracted on dry ice with 80:20 methanol:water, vortexed, centrifugation at 14,000 g at 4 °C for 15 minutes, dried under nitrogen gas, and reconstitution in 35:40:25 acetonitrile:methanol:water for injection. |
Combined analysis:
| Analysis ID | AN003177 |
|---|---|
| Analysis type | MS |
| Chromatography type | Unspecified |
| Chromatography system | Dionex UltiMate 3000 RSLC |
| Column | HILICpak VT50 2D (150 mm x 2.0 mm,5um particle size,Shodex,Japan) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH002349 |
| Chromatography Summary: | Liquid chromatography separation was achieved on a HILICpak VT50 2D column (150 mm x 2.0 mm, 5 µm particle size, Shodex, Japan). Buffer A consists of 90% acetonitrile, 10% water, containing 20 mM Triethylamine : Formic acid at pH 9.18; Buffer B consists of 5% acetonitrile, 95% water containing 54 mM Triethyl-amine : Formic acid at pH 3.03. Flow rate is 0.2mL/min from 0 to 5 minutes, then 0.3 mL/min from 5.1 to 58 min, and reduced again to 0.2mL/min from 58.1 to 60 min. The gradient starts with 0%B from 0 to 10 min, then increases linearly from 0 to 16%B from 10 to 27 min, up to 65%B at 32 min, 87%B at 34 min, 100%B hold from 34.1 to 47 min, then 0%B from 47.1 to 60 min. |
| Instrument Name: | Dionex UltiMate 3000 RSLC |
| Column Name: | HILICpak VT50 2D (150 mm x 2.0 mm,5um particle size,Shodex,Japan) |
| Chromatography Type: | Unspecified |
MS:
| MS ID: | MS002955 |
| Analysis ID: | AN003177 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The Mass Spec parameters are set as Source Fragmentation: None; Sheath gas flow rate: 45; Aux gas flow rate: 15; Sweep gas flow rate: 3; Spray voltage: 3.00 kV; Ca-pillary temp: 310°C; S-lens RF level: 50; Aux gas heater temp: 350°C; For Full MS: Scan range: 65.0 to 975.0 m/z; Resolution: 140,000; Polarity: Negative; AGC target: 3e6; Max-imum IT: 500 ms. |
| Ion Mode: | NEGATIVE |
