#METABOLOMICS WORKBENCH JRabinowitz646_20231114_121259 DATATRACK_ID:4458 STUDY_ID:ST002980 ANALYSIS_ID:AN004898 PROJECT_ID:PR001856 VERSION 1 CREATED_ON November 19, 2023, 6:47 pm #PROJECT PR:PROJECT_TITLE Glycine homeostasis requires reverse SHMT flux PR:PROJECT_TYPE LCMS metabolomics PR:PROJECT_SUMMARY The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly PR:PROJECT_SUMMARY converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such PR:PROJECT_SUMMARY one-carbon unit production plays a critical role in development, the immune PR:PROJECT_SUMMARY system, and cancer. Using rodent models, here we show that the whole-body SHMT PR:PROJECT_SUMMARY flux acts to net consume rather than produce glycine. Pharmacological inhibition PR:PROJECT_SUMMARY of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating PR:PROJECT_SUMMARY glycine levels up to eight-fold. Stable isotope tracing revealed that the liver PR:PROJECT_SUMMARY converts glycine to serine, which is then converted by serine dehydratase into PR:PROJECT_SUMMARY pyruvate and burned in the tricarboxylic acid cycle. In response to diets PR:PROJECT_SUMMARY deficient in serine and glycine, de novo biosynthetic flux was unaltered but PR:PROJECT_SUMMARY SHMT2- and serine dehydratase-mediated catabolic flux was lower. Thus, PR:PROJECT_SUMMARY glucose-derived serine synthesis does not respond to systemic demand. Instead, PR:PROJECT_SUMMARY circulating serine and glycine homeostasis is maintained through variable PR:PROJECT_SUMMARY consumption, with liver SHMT2 a major glycine-consuming enzyme. PR:INSTITUTE Princeton University PR:DEPARTMENT Department of Chemistry PR:LABORATORY Josh Rabinowitz PR:LAST_NAME McBride PR:FIRST_NAME Matthew PR:ADDRESS Carl Icahn Lab, South Drive, Princeton, NJ 08544 PR:EMAIL matthewmcbride@princeton.edu PR:PHONE 8567457389 #STUDY ST:STUDY_TITLE Water soluble metabolomics in mice upon loss of SHMT ST:STUDY_SUMMARY The enzyme SHMT interconverts the amino acids serine and glycine as part of the ST:STUDY_SUMMARY folate cycle. To explore the role of SHMT in amino acid homeostasis, Mice were ST:STUDY_SUMMARY treated with a small molecule inhibitor of SHMT (SHIN2) or had Shmt2 genetically ST:STUDY_SUMMARY knocked-out in a liver specific manner. Serum and liver samples were collected ST:STUDY_SUMMARY and underwent LC-MS metabolomics analysis. ST:INSTITUTE Princeton University ST:LAST_NAME McBride ST:FIRST_NAME Matthew ST:ADDRESS Carl Icahn Lab, South Drive, Princeton, NJ 08544 ST:EMAIL matthewmcbride@princeton.edu ST:PHONE 8567457389 #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 - Veh_1_serum Sample type:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME=Veh_1_serum_NEG.mzXML; RAW_FILE_NAME=Veh_1_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - Veh_2_serum Sample type:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME=Veh_2_serum_NEG.mzXML; RAW_FILE_NAME=Veh_2_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - Veh_3_serum Sample type:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME=Veh_3_serum_NEG.mzXML; RAW_FILE_NAME=Veh_3_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHIN2_1_serum Sample type:Serum | Genotype:Wild-type | Treatment:SHIN2 RAW_FILE_NAME=SHIN2_1_serum_NEG.mzXML; RAW_FILE_NAME=SHIN2_1_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHIN2_2_serum Sample type:Serum | Genotype:Wild-type | Treatment:SHIN2 RAW_FILE_NAME=SHIN2_2_serum_NEG.mzXML; RAW_FILE_NAME=SHIN2_2_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHIN2_3_serum Sample type:Serum | Genotype:Wild-type | Treatment:SHIN2 RAW_FILE_NAME=SHIN2_3_serum_NEG.mzXML; RAW_FILE_NAME=SHIN2_3_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - Veh_1_liver Sample type:Liver | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME=Veh_1_liver_NEG.mzXML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Veh_2_liver Sample type:Liver | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME=Veh_2_liver_NEG.mzXML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Veh_3_liver Sample type:Liver | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME=Veh_3_liver_NEG.mzXML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - SHIN2_1_liver Sample type:Liver | Genotype:Wild-type | Treatment:SHIN2 RAW_FILE_NAME=SHIN2_1_liver_NEG.mzXML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - SHIN2_2_liver Sample type:Liver | Genotype:Wild-type | Treatment:SHIN2 RAW_FILE_NAME=SHIN2_2_liver_NEG.mzXML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - SHIN2_3_liver Sample type:Liver | Genotype:Wild-type | Treatment:SHIN2 RAW_FILE_NAME=SHIN2_3_liver_NEG.mzXML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_15_serum Sample type:Serum | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_15_serum_NEG.mzXML; RAW_FILE_NAME=WT_15_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_20_serum Sample type:Serum | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_20_serum_NEG.mzXML; RAW_FILE_NAME=WT_20_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_34_serum Sample type:Serum | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_34_serum_NEG.mzXML; RAW_FILE_NAME=WT_34_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_37_serum Sample type:Serum | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_37_serum_NEG.mzXML; RAW_FILE_NAME=WT_37_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_46_serum Sample type:Serum | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_46_serum_NEG.mzXML; RAW_FILE_NAME=WT_46_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_21_serum Sample type:Serum | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_21_serum_NEG.mzXML; RAW_FILE_NAME=KO_21_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_22_serum Sample type:Serum | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_22_serum_NEG.mzXML; RAW_FILE_NAME=KO_22_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_23_serum Sample type:Serum | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_23_serum_NEG.mzXML; RAW_FILE_NAME=KO_23_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_35_serum Sample type:Serum | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_35_serum_NEG.mzXML; RAW_FILE_NAME=KO_35_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_43_serum Sample type:Serum | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_43_serum_NEG.mzXML; RAW_FILE_NAME=KO_43_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_44_serum Sample type:Serum | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_44_serum_NEG.mzXML; RAW_FILE_NAME=KO_44_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_45_serum Sample type:Serum | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_45_serum_NEG.mzXML; RAW_FILE_NAME=KO_45_serum_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_15_liver Sample type:Liver | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_15_liver_NEG.mzXML; RAW_FILE_NAME=WT_15_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_20_liver Sample type:Liver | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_20_liver_NEG.mzXML; RAW_FILE_NAME=WT_20_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_34_liver Sample type:Liver | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_34_liver_NEG.mzXML; RAW_FILE_NAME=WT_34_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_37_liver Sample type:Liver | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_37_liver_NEG.mzXML; RAW_FILE_NAME=WT_37_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_WT_46_liver Sample type:Liver | Genotype:Wild-type | Treatment:None RAW_FILE_NAME=WT_46_liver_NEG.mzXML; RAW_FILE_NAME=WT_46_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_21_liver Sample type:Liver | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_21_liver_NEG.mzXML; RAW_FILE_NAME=KO_21_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_22_liver Sample type:Liver | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_22_liver_NEG.mzXML; RAW_FILE_NAME=KO_22_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_23_liver Sample type:Liver | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_23_liver_NEG.mzXML; RAW_FILE_NAME=KO_23_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_35_liver Sample type:Liver | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_35_liver_NEG.mzXML; RAW_FILE_NAME=KO_35_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_43_liver Sample type:Liver | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_43_liver_NEG.mzXML; RAW_FILE_NAME=KO_43_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_44_liver Sample type:Liver | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_44_liver_NEG.mzXML; RAW_FILE_NAME=KO_44_liver_POS.mzXML SUBJECT_SAMPLE_FACTORS - SHMT2flox_KO_45_liver Sample type:Liver | Genotype:Liver KO | Treatment:None RAW_FILE_NAME=KO_45_liver_NEG.mzXML; RAW_FILE_NAME=KO_45_liver_POS.mzXML #COLLECTION CO:COLLECTION_SUMMARY For pharmacological inhibition of SHMT, mice were treated with Vehicle or SHIN2 CO:COLLECTION_SUMMARY for 12 hours and then blood serum and liver samples were collected. For genetic CO:COLLECTION_SUMMARY loss of Shmt2, blood serum and liver samples were collected from mice 21 days CO:COLLECTION_SUMMARY after liver-specific gene knockout. CO:SAMPLE_TYPE Blood (serum) and Liver #TREATMENT TR:TREATMENT_SUMMARY For pharmacological inhibition of SHMT, mice (C57BL/6N) were divided into two TR:TREATMENT_SUMMARY groups and received Vehicle (20% 2-hydroxypropyl-β-cyclodextrin) or SHIN2 (200 TR:TREATMENT_SUMMARY mg/kg) for 12 hours. For genetic loss of Shmt2, Shmt2(flox/flox) mice and TR:TREATMENT_SUMMARY wild-type litter mate controls were injected with AAV8-TBG-Cre viral particles TR:TREATMENT_SUMMARY to induce liver-specific gene knockout and samples were harvested 21 days later. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Water soluble metabolites were extracted from serum and liver samples. 3ul of SP:SAMPLEPREP_SUMMARY serum was extracted with 120 ul (40X) of 100% methanol, cooled on ice for 10 SP:SAMPLEPREP_SUMMARY minutes, centrifuged at 16,000 x g for 30 minutes, and supernatant collected. SP:SAMPLEPREP_SUMMARY 20mg of ground liver tissue was extracted with 800ul of 40:40:20 SP:SAMPLEPREP_SUMMARY methanol:acetonitrile:water, cooled on ice for 10 minutes, centrifuged at 16,000 SP:SAMPLEPREP_SUMMARY x g for 30 minutes, and supernatant collected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters XBridge BEH Amide (100 x 2.1mm,2.5um) CH:SOLVENT_A 95% water/5% acetonitrile with 20 mM ammonium acetate, 20 mM ammonium hydroxide, CH:SOLVENT_A pH 9.4 CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0 minutes, 85% B; 2 minutes, 85% B; 3 minutes, 80% B; 5 minutes, 80% B; 6 CH:FLOW_GRADIENT minutes, 75% B; 7 minutes, 75% B; 8 minutes, 70% B; 9 minutes, 70% B; 10 CH:FLOW_GRADIENT minutes, 50% B; 12 minutes, 50% B; 13 minutes, 25% B; 16 minutes, 25% B; 18 CH:FLOW_GRADIENT minutes, 0% B; 23 minutes, 0% B; 24 minutes, 85% B; 30 minutes, 85% B CH:FLOW_RATE 150 μl/min CH:COLUMN_TEMPERATURE 25°C #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 POSITIVE MS:MS_COMMENTS MS full scans were in positive ion mode with a resolution of 140,000 at m/z 200 MS:MS_COMMENTS and scan range of 70–1,000 m/z. The automatic gain control (AGC) target was 1 MS:MS_COMMENTS × 10^6. LC-MS peak files were analyzed and visualized with El-MAVEN (Elucidata) MS:MS_COMMENTS using 5 ppm ion extraction window, minimum peak intensity of 1 x 10^5 ions, and MS:MS_COMMENTS minimum signal to background blank ratio of 2. MS:MS_RESULTS_FILE ST002980_AN004898_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END