#METABOLOMICS WORKBENCH borjalanzon_20191219_092327 DATATRACK_ID:1879 STUDY_ID:ST001303 ANALYSIS_ID:AN002169 PROJECT_ID:PR000884 VERSION 1 CREATED_ON January 9, 2020, 1:15 pm #PROJECT PR:PROJECT_TITLE TGFβ3 heterozygous mice PR:PROJECT_TYPE Mice nephropathy in lipotoxic model PR:PROJECT_SUMMARY Transforming growth factor β (TGFβ) family comprises the main player in the PR:PROJECT_SUMMARY development of fibrosis including three isoforms: TGFβ1, TGFβ2 and TGFβ3. PR:PROJECT_SUMMARY TGFβ3 may play an antifibrotic role at the renal level, counteracting the role PR:PROJECT_SUMMARY of TGFβ1, using a mouse model heterozygous for the TGFβ3 gene (TGFβ3+/-). PR:PROJECT_SUMMARY Partial deletion of TGFβ3 causes in the mice albuminuria, loss of glomerular PR:PROJECT_SUMMARY filtration rate, accelerated fibrosis, epithelial-to-mesenchymal transition and PR:PROJECT_SUMMARY increment of glomerular basement membrane thickening. PR:INSTITUTE University Rey Juan Carlos PR:DEPARTMENT Basics Science of Health PR:LAST_NAME Lanzon PR:FIRST_NAME Borja PR:ADDRESS Avenida de Atenas S/N, Alcorcón, Madrid, 28922, Spain PR:EMAIL borja.lanzon@urjc.es PR:PHONE 663692554 #STUDY ST:STUDY_TITLE TGF-Beta 3 heterozygous mice ST:STUDY_TYPE Mice nephropathy in lipotoxic model ST:STUDY_SUMMARY Transforming growth factor β (TGFβ) family comprises the main player in the ST:STUDY_SUMMARY development of fibrosis including three isoforms: TGFβ1, TGFβ2 and TGFβ3. ST:STUDY_SUMMARY TGFβ3 may play an antifibrotic role at the renal level, counteracting the role ST:STUDY_SUMMARY of TGFβ1, using a mouse model heterozygous for the TGFβ3 gene (TGFβ3+/-). ST:STUDY_SUMMARY Partial deletion of TGFβ3 causes in the mice albuminuria, loss of glomerular ST:STUDY_SUMMARY filtration rate, accelerated fibrosis, epithelial-to-mesenchymal transition and ST:STUDY_SUMMARY increment of glomerular basement membrane thickening. ST:INSTITUTE University Rey Juan Carlos ST:DEPARTMENT Basics Science of Health ST:LAST_NAME Lanzon ST:FIRST_NAME Borja ST:ADDRESS Avenida de Atenas S/N ST:EMAIL borja.lanzon@urjc.es ST:PHONE 663692554 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 14 ST:NUM_MALES 14 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN c57bl6 SU:AGE_OR_AGE_RANGE 16 weeks SU:GENDER Male #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 - 269 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 269 SUBJECT_SAMPLE_FACTORS - 119 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 119 SUBJECT_SAMPLE_FACTORS - 267 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 267 SUBJECT_SAMPLE_FACTORS - 130 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 130 SUBJECT_SAMPLE_FACTORS - 127 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 127 SUBJECT_SAMPLE_FACTORS - 98 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 98 SUBJECT_SAMPLE_FACTORS - 25 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 25 SUBJECT_SAMPLE_FACTORS - 132 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 132 SUBJECT_SAMPLE_FACTORS - 251 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 251 SUBJECT_SAMPLE_FACTORS - 120 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 120 SUBJECT_SAMPLE_FACTORS - 79 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 79 SUBJECT_SAMPLE_FACTORS - 129 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 129 SUBJECT_SAMPLE_FACTORS - 128 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 128 SUBJECT_SAMPLE_FACTORS - 92 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 92 SUBJECT_SAMPLE_FACTORS - QC1 Genotype:QC RAW_FILE_NAME=QC1 SUBJECT_SAMPLE_FACTORS - QC2 Genotype:QC RAW_FILE_NAME=QC2 SUBJECT_SAMPLE_FACTORS - QC3 Genotype:QC RAW_FILE_NAME=QC3 SUBJECT_SAMPLE_FACTORS - QC4 Genotype:QC RAW_FILE_NAME=QC4 SUBJECT_SAMPLE_FACTORS - QC5 Genotype:QC RAW_FILE_NAME=QC5 #COLLECTION CO:COLLECTION_SUMMARY Kidney samples were powdered with mortar and pestle. Method used for extraction CO:COLLECTION_SUMMARY was previously validated for tissue CO:SAMPLE_TYPE Kidney CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Kidney homogenate was prepared by adding cold (−20 °C) methanol/water (1:1, TR:TREATMENT_SUMMARY v/v), (1:10 tissue/solvent). Tissue disruption was achieved with Tissue- Lyser TR:TREATMENT_SUMMARY LT homogenizer (Qiagen, Germany) for metabolite extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 100 μL of kidney tissue homogenate was vortex-mixed with 320 μL of methanol SP:SAMPLEPREP_SUMMARY for 2 min, followed by the addition of 80 μL of MTBE for the extraction of SP:SAMPLEPREP_SUMMARY nonpolar compounds. Then, vials were rapidly capped and placed on a shaker for 1 SP:SAMPLEPREP_SUMMARY h at room temperature. The extracted samples were centrifuged at 4000g for 20 SP:SAMPLEPREP_SUMMARY min at 20 °C. For GC−MS analysis, 300 μL of supernatant was evaporated to SP:SAMPLEPREP_SUMMARY dryness (SpeedVac Concentrator System, Thermo Fisher Scientific, Waltham, MA). SP:SAMPLEPREP_SUMMARY Methoxymation was then performed with 20 μL of 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 h. For silylation, 20 μL of SP:SAMPLEPREP_SUMMARY BSTFA/TMCS (99:1) was added and 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 tricosane (20 ppm) as internal standard (IS) was added to each vial prior to SP:SAMPLEPREP_SUMMARY injection. For LC−MS analysis, 90 μL of supernatant was transferred to an SP:SAMPLEPREP_SUMMARY ultra-high-performance liquid chromatography−mass spectrometry. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY LC-MS (+) CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Poroshell 120 EC-C8 (100 x 2.1mm, 2.5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS A UHPLC system (1290 Infinity UHPLC system, Agilent Technologies, Waldbronn, MS:MS_COMMENTS Germany), consisting of two degassers, two binary pumps, and a thermostated MS:MS_COMMENTS autosampler (maintained at 4°C) coupled with 6545 QTOF MS detector, was used in MS:MS_COMMENTS positive and negative ESI modes. In brief, 1 μL of each sample was injected MS:MS_COMMENTS into a reverse-phase Zorbax Eclipse Plus C8 column, 2.1 × 150 mm; 1.8 μm MS:MS_COMMENTS (Agilent Technologies) thermostated at 60°C. The gradient used for the analysis MS:MS_COMMENTS consisted of a mobile phase A (10 mM ammonium formate in Milli-Q water) and MS:MS_COMMENTS mobile phase B (10 mM ammonium formate in methanol:isopropanol, 85:15) pumped at MS:MS_COMMENTS 0.5 mL/min. The chromatography gradient started at 82% phase B, increasing to MS:MS_COMMENTS 90% B in 17 min. The gradient then increased to 100% B by minute 18 and was MS:MS_COMMENTS maintained for 2 minutes until 20 min. The starting condition was returned to by MS:MS_COMMENTS 21.5 min, followed by an 8.5 min reequilibration time, taking the total run time MS:MS_COMMENTS to 30 min. Data were collected in full scan mode from 100 to 1200 m/z, with a MS:MS_COMMENTS scan rate of 1.02 scans/s. The capillary voltage was set to 3500 V; the drying MS:MS_COMMENTS gas flow rate was 12 L/min at 290°C and gas nebulizer 45 psi, fragmentor MS:MS_COMMENTS voltage 175 V, and octopole radio frequency voltage (OCT RF Vpp) 750 V. Two MS:MS_COMMENTS reference masses were used over the course of the whole analysis: m/z 121.0509 MS:MS_COMMENTS (protonated purine) and m/z 922.0098 (protonated hexakis, MS:MS_COMMENTS (1H,1H,3H-tetrafluoropropoxy)phosphazine (HP-921)). These masses were MS:MS_COMMENTS continuously infused into the system to provide constant mass correction. MS:MS_COMMENTS Samples were randomly analyzed throughout the run. MS:MS_RESULTS_FILE ST001303_AN002169_Results.txt UNITS:Area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END