#METABOLOMICS WORKBENCH mclasquin_20211022_133843 DATATRACK_ID:2900 STUDY_ID:ST001952 ANALYSIS_ID:AN003177 PROJECT_ID:PR001239 VERSION 1 CREATED_ON October 26, 2021, 10:10 am #PROJECT PR:PROJECT_TITLE GLS2KO vs WT mouse hepatocytes PR:PROJECT_TYPE Genotype PR:PROJECT_SUMMARY Test the effect of knocking out GLS2 in primary mouse heptocytes. This data set PR:PROJECT_SUMMARY was generated by applying Mixed Mode chromatography coupled to a Q Exactive Plus PR:PROJECT_SUMMARY Orbitrap Mass Spectrometer with enhanced MS resolution up to 280,000. The most PR:PROJECT_SUMMARY significantly altered metabolites as identified by XCMS were used to test the PR:PROJECT_SUMMARY ability of a Message Passing Neural Network (MPNN) Model to rank order PR:PROJECT_SUMMARY metabolite IDs. PR:INSTITUTE Pfizer PR:DEPARTMENT Internal Medicine Research Unit, WRDM PR:LAST_NAME Clasquin PR:FIRST_NAME Michelle PR:ADDRESS 1 Portland St., Cambridge, MA 02139 PR:EMAIL michelle.clasquin@pfizer.com PR:PHONE 6174487289 PR:CONTRIBUTORS Gang Xing, Yuji Shi #STUDY ST:STUDY_TITLE GLS2KO vs WT mouse hepatocytes ST:STUDY_TYPE Genotype ST:STUDY_SUMMARY Test the effect of GLS2 knockout in primary mouse hepatocytes. We isolated ST:STUDY_SUMMARY hepatocytes from GLS2 knockout and wild-type mice, and briefly applied media ST:STUDY_SUMMARY lacking L-glutamine (2 hours). Sixty minutes after resupplying Gln, metabolites ST:STUDY_SUMMARY were extracted and analyzed with the Mixed Mode method. Data were processed ST:STUDY_SUMMARY through XCMS, and features were filtered for p<0.01, fold change >2, and a ST:STUDY_SUMMARY minimum intensity of 1x10^6. Sorting by smallest p value, the first extracted ST:STUDY_SUMMARY ion chromatogram (EIC) with good chromatographic peak shape corresponded to ST:STUDY_SUMMARY 188.0567m/z at 24.41 min. Six putative IDs were within 3ppm of the ST:STUDY_SUMMARY experimentally observed m/z, representing two chemical formulas, none of which ST:STUDY_SUMMARY had documented retention times in training or test sets. Amongst these potential ST:STUDY_SUMMARY IDs, the Message Passing Neural Network (MPNN) model correctly predicted ST:STUDY_SUMMARY N-acetyl-L-glutamic acid as the most likely candidate, as verified by injection ST:STUDY_SUMMARY of purchased standards. The next most significant difference between GLS2KO vs ST:STUDY_SUMMARY WT was 117.0196m/z observed at 20.07 minutes. The model had been trained on 2/6 ST:STUDY_SUMMARY of the putative IDs. Despite the four additional isomers suggested, the model ST:STUDY_SUMMARY correctly selected succinate as reduced by GLS2 KO (Figure 5B). The third most ST:STUDY_SUMMARY significant hit corresponds to 171.0068m/z at 23.11 min. Although glycerol 1-P ST:STUDY_SUMMARY and 2-P are both potential hits, almost indistinguishable by the model, the ST:STUDY_SUMMARY large gap in retention times between these top hits and the Cl- adducts of ST:STUDY_SUMMARY threonate (+ isomers) is apparent, further supporting the correct identification ST:STUDY_SUMMARY as glycerol mono-phosphate. Expansion of the list to include p<0.05 leads to the ST:STUDY_SUMMARY identification of Glutamine, Glutamate, and other downstream metabolites known ST:STUDY_SUMMARY to be altered by GLS2 KO. ST:INSTITUTE Pfizer ST:LAST_NAME Clasquin ST:FIRST_NAME Michelle ST:ADDRESS 1 Portland St., Cambridge, MA 02139 ST:EMAIL michelle.clasquin@pfizer.com ST:PHONE 6174487289 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 6 ST:NUM_MALES 6 #SUBJECT SU:SUBJECT_TYPE Mammal 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 - 1 Genotype:Wild-type RAW_FILE_NAME=DS-181015-00034_GLS2_33_GLS2-WT_Glutamine_60min_GC9 SUBJECT_SAMPLE_FACTORS - 2 Genotype:Wild-type RAW_FILE_NAME=DS-181015-00033_GLS2_32_GLS2-WT_Glutamine_60min_GC8 SUBJECT_SAMPLE_FACTORS - 3 Genotype:Wild-type RAW_FILE_NAME=DS-181015-00032_GLS2_31_GLS2-WT_Glutamine_60min_GC7 SUBJECT_SAMPLE_FACTORS - 4 Genotype:GLS2 KO RAW_FILE_NAME=DS-181015-00040_GLS2_39_GLS2-KO_Glutamine_60min_GD3 SUBJECT_SAMPLE_FACTORS - 5 Genotype:GLS2 KO RAW_FILE_NAME=DS-181015-00039_GLS2_38_GLS2-KO_Glutamine_60min_GD2 SUBJECT_SAMPLE_FACTORS - 6 Genotype:GLS2 KO RAW_FILE_NAME=DS-181015-00038_GLS2_37_GLS2-KO_Glutamine_60min_GD1 #COLLECTION CO:COLLECTION_SUMMARY All samples extracted from mice were collected in accordance with regulations CO:COLLECTION_SUMMARY and established guidelines for humane treatment of research animals and were CO:COLLECTION_SUMMARY reviewed and approved by an Institutional Animal Care and Use Committee, Project CO:COLLECTION_SUMMARY Code HD-EX00066. Primary hepatocytes were isolated from male mice between 12-18 CO:COLLECTION_SUMMARY weeks of age by the two-step collagenase perfusion method. Mice were fasted 16 CO:COLLECTION_SUMMARY hours before the ex-periments. After isolation, cells were plated in M199 media CO:COLLECTION_SUMMARY with 10% FBS for 4 hours in 6-well plates pre-coated with collagen I. After CO:COLLECTION_SUMMARY cells attached to the plates, they were washed with glucose output media (GOM) CO:COLLECTION_SUMMARY (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 20 mM CO:COLLECTION_SUMMARY NaCO3, 25 mM HEPES pH 7.4, and 0.025% BSA), and incubated in fresh GOM for 2 CO:COLLECTION_SUMMARY hour. GOM media was replaced with fresh, pre-warmed GOM media and cellular CO:COLLECTION_SUMMARY treatments initiated. Hepatocytes were treated with 5mM unlabeled glutamine for CO:COLLECTION_SUMMARY 60 min. Hepatocytes were washed with ice cold PBS twice and immediately frozen CO:COLLECTION_SUMMARY in liquid nitrogen. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY All samples extracted from mice were collected in accordance with regulations TR:TREATMENT_SUMMARY and established guidelines for humane treatment of research animals and were TR:TREATMENT_SUMMARY reviewed and approved by an Institutional Animal Care and Use Committee, Project TR:TREATMENT_SUMMARY Code HD-EX00066. Primary hepatocytes were isolated from male mice between 12-18 TR:TREATMENT_SUMMARY weeks of age by the two-step collagenase perfusion method. Mice were fasted 16 TR:TREATMENT_SUMMARY hours before the ex-periments. After isolation, cells were plated in M199 media TR:TREATMENT_SUMMARY with 10% FBS for 4 hours in 6-well plates pre-coated with collagen I. After TR:TREATMENT_SUMMARY cells attached to the plates, they were washed with glucose output media (GOM) TR:TREATMENT_SUMMARY (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 20 mM TR:TREATMENT_SUMMARY NaCO3, 25 mM HEPES pH 7.4, and 0.025% BSA), and incubated in fresh GOM for 2 TR:TREATMENT_SUMMARY hour. GOM media was replaced with fresh, pre-warmed GOM media and cellular TR:TREATMENT_SUMMARY treatments initiated. Hepatocytes were treated with 5mM unlabeled glutamine for TR:TREATMENT_SUMMARY 60 min. Hepatocytes were washed with ice cold PBS twice and immediately frozen TR:TREATMENT_SUMMARY in liquid nitrogen. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted on dry ice with 80:20 methanol:water, vortexed, SP:SAMPLEPREP_SUMMARY centrifugation at 14,000 g at 4 °C for 15 minutes, dried under nitrogen gas, SP:SAMPLEPREP_SUMMARY and reconstitution in 35:40:25 acetonitrile:methanol:water for injection. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Liquid chromatography separation was achieved on a HILICpak VT50 2D column (150 CH:CHROMATOGRAPHY_SUMMARY mm x 2.0 mm, 5 µm particle size, Shodex, Japan). Buffer A consists of 90% CH:CHROMATOGRAPHY_SUMMARY acetonitrile, 10% water, containing 20 mM Triethylamine : Formic acid at pH CH:CHROMATOGRAPHY_SUMMARY 9.18; Buffer B consists of 5% acetonitrile, 95% water containing 54 mM CH:CHROMATOGRAPHY_SUMMARY Triethyl-amine : Formic acid at pH 3.03. Flow rate is 0.2mL/min from 0 to 5 CH:CHROMATOGRAPHY_SUMMARY minutes, then 0.3 mL/min from 5.1 to 58 min, and reduced again to 0.2mL/min from CH:CHROMATOGRAPHY_SUMMARY 58.1 to 60 min. The gradient starts with 0%B from 0 to 10 min, then increases CH:CHROMATOGRAPHY_SUMMARY linearly from 0 to 16%B from 10 to 27 min, up to 65%B at 32 min, 87%B at 34 min, CH:CHROMATOGRAPHY_SUMMARY 100%B hold from 34.1 to 47 min, then 0%B from 47.1 to 60 min. CH:CHROMATOGRAPHY_TYPE Other CH:INSTRUMENT_NAME Dionex UltiMate 3000 RSLC CH:COLUMN_NAME HILICpak VT50 2D column (150 mm x 2.0 mm, 5 µm particle size, Shodex, Japan) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The Mass Spec parameters are set as Source Fragmentation: None; Sheath gas flow MS:MS_COMMENTS rate: 45; Aux gas flow rate: 15; Sweep gas flow rate: 3; Spray voltage: 3.00 kV; MS:MS_COMMENTS Ca-pillary temp: 310°C; S-lens RF level: 50; Aux gas heater temp: 350°C; For MS:MS_COMMENTS Full MS: Scan range: 65.0 to 975.0 m/z; Resolution: 140,000; Polarity: Negative; MS:MS_COMMENTS AGC target: 3e6; Max-imum IT: 500 ms. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples 1 2 3 4 5 6 Factors Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:GLS2 KO Genotype:GLS2 KO Genotype:GLS2 KO Succinic acid 1080807 1178016 991949.1 254526.6 314118.3 253132.1 N-acetyl-L-glutamic acid 3483933 3906550 3732787 535850.6 513000.2 537080.9 D-glycerol 1-phosphate 1565587 1994412 1967104 3460778 2645520 3765885 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Median RT(min) median Mz HMDB KEGG ID PUbChem ID Succinic acid 19.97475 117.0195 HMDB0000254 C00042 1738118 N-acetyl-L-glutamic acid 24.38281 188.0567 HMDB0001138 C00624 70914 D-glycerol 1-phosphate 23.10928 171.0066 HMDB0000126 C00093 439162 METABOLITES_END #END