#METABOLOMICS WORKBENCH Carol_Glez_20181221_084352 DATATRACK_ID:1589 STUDY_ID:ST001117 ANALYSIS_ID:AN001815 PROJECT_ID:PR000748 VERSION 1 CREATED_ON December 26, 2018, 12:51 pm #PROJECT PR:PROJECT_TITLE A Metabolomic study of hibernating Syrian hamster brain: in search of PR:PROJECT_TITLE neuroprotective agents PR:PROJECT_SUMMARY hamster brain samples, divided in 3 groups: torpor, arousal, control group were PR:PROJECT_SUMMARY compared via metabolomics analysis PR:INSTITUTE Universidad CEU San Pablo PR:LAST_NAME Gonzalez-Riano PR:FIRST_NAME Carolina PR:ADDRESS Facultad de Farmacia, Universidad CEU San Pablo, Campus Monteprincipe, Boadilla PR:ADDRESS del Monte, Boadilla del Monte, Madrid, 28668, Spain PR:EMAIL car.gonzalez@ceindo.ceu.es PR:PHONE 00 34 91 3724753 #STUDY ST:STUDY_TITLE A Metabolomic study of hibernating Syrian hamster brain: in search of ST:STUDY_TITLE neuroprotective agents ST:STUDY_TYPE Multiplatform non-targeted metabolomics ST:STUDY_SUMMARY hamster brain samples, divided in 3 groups: torpor, arousal, control group were ST:STUDY_SUMMARY compared via metabolomics analysis ST:INSTITUTE Universidad CEU San Pablo ST:LABORATORY CEMBIO (Centre for Metabolomics and Bioanalysis) ST:LAST_NAME Gonzalez-Riano ST:FIRST_NAME Carolina ST:ADDRESS Facultad de Farmacia, Universidad CEU San Pablo, Campus Monteprincipe, Boadilla ST:ADDRESS del Monte, Boadilla del Monte, Madrid, 28668, Spain ST:EMAIL car.gonzalez@ceindo.ceu.es ST:PHONE 00 34 91 3724753 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mesocricetus Auratus SU:TAXONOMY_ID 10036 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - A1 hibernation:arousal SUBJECT_SAMPLE_FACTORS - A3 hibernation:arousal SUBJECT_SAMPLE_FACTORS - A6 hibernation:arousal SUBJECT_SAMPLE_FACTORS - A10 hibernation:arousal SUBJECT_SAMPLE_FACTORS - A11 hibernation:arousal SUBJECT_SAMPLE_FACTORS - C1 hibernation:control SUBJECT_SAMPLE_FACTORS - C2 hibernation:control SUBJECT_SAMPLE_FACTORS - C3 hibernation:control SUBJECT_SAMPLE_FACTORS - C4 hibernation:control SUBJECT_SAMPLE_FACTORS - C5 hibernation:control SUBJECT_SAMPLE_FACTORS - T2 hibernation:torpor SUBJECT_SAMPLE_FACTORS - T4 hibernation:torpor SUBJECT_SAMPLE_FACTORS - T5 hibernation:torpor SUBJECT_SAMPLE_FACTORS - T10 hibernation:torpor SUBJECT_SAMPLE_FACTORS - T11 hibernation:torpor #COLLECTION CO:COLLECTION_SUMMARY A total of 15 male 3-month-old Syrian hamsters had free access to food and water CO:COLLECTION_SUMMARY and were kept at 23C with an 8:16-h light/dark cycle for a four to six-week CO:COLLECTION_SUMMARY acclimatization period in our animal facility. All animals were euthanized by CO:COLLECTION_SUMMARY decapitation. Brains were then removed and immediately transferred to a CO:COLLECTION_SUMMARY N2(l)-containing recipient to freeze the tissues. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY the whole right hemisphere (300 mg approx.) was analyzed to decrease possible TR:TREATMENT_SUMMARY biological variability due to the brain region employed. Brain homogenate was TR:TREATMENT_SUMMARY prepared by adding cold (-20°C) methanol:water (1:1, v/v), (1:10 TR:TREATMENT_SUMMARY tissue:solvent). Tissue disruption was achieved with TissueLyser LT homogenizer TR:TREATMENT_SUMMARY (Qiagen, Germany) for metabolite extraction. Subsequently, 100 μL of brain TR:TREATMENT_SUMMARY tissue homogenate was vortex-mixed with 320 μL of methanol for 2 min, followed TR:TREATMENT_SUMMARY by the addition of 80 μL of MTBE for the extraction of non-polar compounds. TR:TREATMENT_SUMMARY Then, vials were rapidly capped and placed on a shaker for 1 h at room TR:TREATMENT_SUMMARY temperature. The extracted samples were centrifuged at 4000 g for 20 min at TR:TREATMENT_SUMMARY 20°C. For GC-MS analysis, 300 μL of supernatant was evaporated to dryness TR:TREATMENT_SUMMARY (SpeedVac Concentrator System, Thermo Fisher Scientific, Waltham, MA, USA). TR:TREATMENT_SUMMARY Methoxymation was then performed with 20 µL O-methoxyamine hydrochloride (15 TR:TREATMENT_SUMMARY mg/mL in pyridine) and vigorously vortex-mixed for 5 min. Vials were then TR:TREATMENT_SUMMARY incubated in darkness at room temperature for 16 hours. For silylation, 20 μL TR:TREATMENT_SUMMARY of BSTFA:TMCS (99:1) was added, vortex-mixed for 5 min, and capped vials were TR:TREATMENT_SUMMARY placed in the oven at 70°C for 1 h. Finally, 100 μL of heptane containing TR:TREATMENT_SUMMARY C18:0 methyl ester (10 ppm) as Internal Standard (IS) was added to each vial TR:TREATMENT_SUMMARY prior to injection. For LC-MS analysis, 90 μL of supernatant was transferred to TR:TREATMENT_SUMMARY an Ultra-High Performance Liquid Chromatography-Mass (UHPLC-MS) chromatography TR:TREATMENT_SUMMARY vial with insert and was directly injected into the system. For CE-MS analysis, TR:TREATMENT_SUMMARY 200 μL of initial brain homogenate was centrifuged separately at 16000 g for 30 TR:TREATMENT_SUMMARY min at 15°C. 150 μL of supernatant was evaporated to dryness using the TR:TREATMENT_SUMMARY SpeedVac, and re-suspended in 150 μL of 0.2 mM Methionine Sulfone (IS) in 0.1 M TR:TREATMENT_SUMMARY formic acid. Samples were vortex-mixed, sonicated, and then centrifuged at 16000 TR:TREATMENT_SUMMARY g for 20 min at 4°C. Finally, 100 μL of supernatant was transferred to a CE-MS TR:TREATMENT_SUMMARY vial for the analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY the whole right hemisphere (300 mg approx.) was analyzed to decrease possible SP:SAMPLEPREP_SUMMARY biological variability due to the brain region employed. Brain homogenate was SP:SAMPLEPREP_SUMMARY prepared by adding cold (-20°C) methanol:water (1:1, v/v), (1:10 SP:SAMPLEPREP_SUMMARY tissue:solvent). Tissue disruption was achieved with TissueLyser LT homogenizer SP:SAMPLEPREP_SUMMARY (Qiagen, Germany) for metabolite extraction. Subsequently, 100 μL of brain SP:SAMPLEPREP_SUMMARY tissue homogenate was vortex-mixed with 320 μL of methanol for 2 min, followed SP:SAMPLEPREP_SUMMARY by the addition of 80 μL of MTBE for the extraction of non-polar compounds. SP:SAMPLEPREP_SUMMARY Then, vials were rapidly capped and placed on a shaker for 1 h at room SP:SAMPLEPREP_SUMMARY temperature. The extracted samples were centrifuged at 4000 g for 20 min at SP:SAMPLEPREP_SUMMARY 20°C. For GC-MS analysis, 300 μL of supernatant was evaporated to dryness SP:SAMPLEPREP_SUMMARY (SpeedVac Concentrator System, Thermo Fisher Scientific, Waltham, MA, USA). SP:SAMPLEPREP_SUMMARY Methoxymation was then performed with 20 µL O-methoxyamine hydrochloride (15 SP:SAMPLEPREP_SUMMARY mg/mL in pyridine) and vigorously vortex-mixed for 5 min. Vials were then SP:SAMPLEPREP_SUMMARY incubated in darkness at room temperature for 16 hours. For silylation, 20 μL SP:SAMPLEPREP_SUMMARY of BSTFA:TMCS (99:1) was added, vortex-mixed for 5 min, and capped vials were SP:SAMPLEPREP_SUMMARY placed in the oven at 70°C for 1 h. Finally, 100 μL of heptane containing SP:SAMPLEPREP_SUMMARY C18:0 methyl ester (10 ppm) as Internal Standard (IS) was added to each vial SP:SAMPLEPREP_SUMMARY prior to injection. For LC-MS analysis, 90 μL of supernatant was transferred to SP:SAMPLEPREP_SUMMARY an Ultra-High Performance Liquid Chromatography-Mass (UHPLC-MS) chromatography SP:SAMPLEPREP_SUMMARY vial with insert and was directly injected into the system. For CE-MS analysis, SP:SAMPLEPREP_SUMMARY 200 μL of initial brain homogenate was centrifuged separately at 16000 g for 30 SP:SAMPLEPREP_SUMMARY min at 15°C. 150 μL of supernatant was evaporated to dryness using the SP:SAMPLEPREP_SUMMARY SpeedVac, and re-suspended in 150 μL of 0.2 mM Methionine Sulfone (IS) in 0.1 M SP:SAMPLEPREP_SUMMARY formic acid. Samples were vortex-mixed, sonicated, and then centrifuged at 16000 SP:SAMPLEPREP_SUMMARY g for 20 min at 4°C. Finally, 100 μL of supernatant was transferred to a CE-MS SP:SAMPLEPREP_SUMMARY vial for the analysis #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE CE CH:INSTRUMENT_NAME Agilent 7100 CE CH:COLUMN_NAME Fused-silica capillary from Agilent Technologies #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6224 TOF MS:INSTRUMENT_TYPE TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST001117_AN001815_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END