#METABOLOMICS WORKBENCH chanpearl_20231026_020405 DATATRACK_ID:4418 STUDY_ID:ST002947 ANALYSIS_ID:AN004832 PROJECT_ID:PR001832 VERSION 1 CREATED_ON October 29, 2023, 9:39 am #PROJECT PR:PROJECT_TITLE A study of the physiological functions and impact of secretory protein Cgref1 PR:PROJECT_SUMMARY Cell Growth Regulator with EF-Hand Domain 1 (Cgref1) is a secretory protein with PR:PROJECT_SUMMARY limited information on its functions. Our group has performed an extensive study PR:PROJECT_SUMMARY using both in-vitro and in-vivo models. Particularly, we used transgenic mice in PR:PROJECT_SUMMARY which the Cgref1 gene is deleted to enable loss-of-function studies. PR:PROJECT_SUMMARY Cgref1-knockout (KO) mice are generally leaner and metabolically healthier PR:PROJECT_SUMMARY compared to wild type mice. To gain evidence of certain parameters, metabolomics PR:PROJECT_SUMMARY studies have been performed for this project. PR:INSTITUTE The University of Hong Kong PR:DEPARTMENT School of Biomedical Sciences PR:LABORATORY L3-53 PR:LAST_NAME Chan PR:FIRST_NAME Pearl PR:ADDRESS 21 Sassoon Road, Pokfulam, Hong Kong, HKSAR, NA, Hong Kong PR:EMAIL pearl20@connect.hku.hk PR:PHONE +85239176812 #STUDY ST:STUDY_TITLE Hepatic lipid profiles of wild type and Cgref1-depleted mice of C57BL/6 strain ST:STUDY_TITLE obtained by an untargeted LC-MS/MS analysis ST:STUDY_SUMMARY Among the range of experiments and observations recorded, we compared the lipid ST:STUDY_SUMMARY expression in the liver tissues between wild type and Cgref1-knockout mice of ST:STUDY_SUMMARY C57BL/6 strain using untargeted LC-MS/MS (n=3 per group). Particularly, the ST:STUDY_SUMMARY results revealed that Cgref1-knockout (KO) mice overall have lower levels of ST:STUDY_SUMMARY triglyceride and diglyceride species. Such finding provides supportive evidence ST:STUDY_SUMMARY that Cgref1 may promote de novo lipogenesis in the liver and increase the risk ST:STUDY_SUMMARY of fatty liver development. ST:INSTITUTE The University of Hong Kong ST:DEPARTMENT School of Biomedical Sciences ST:LABORATORY L3-53 ST:LAST_NAME Chan ST:FIRST_NAME Pearl ST:ADDRESS 21 Sassoon Road, Pokfulam, Hong Kong ST:EMAIL pearl20@connect.hku.hk ST:PHONE +85239176812 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #FACTORS #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 - WT1 Genotype:Wild-type RAW_FILE_NAME=20230329-lipid-WT1.raw SUBJECT_SAMPLE_FACTORS - WT2 Genotype:Wild-type RAW_FILE_NAME=20230329-lipid-WT2.raw SUBJECT_SAMPLE_FACTORS - WT3 Genotype:Wild-type RAW_FILE_NAME=20230329-lipid-WT3.raw SUBJECT_SAMPLE_FACTORS - CG1 Genotype:Cgref1-knockout RAW_FILE_NAME=20230329-lipid-CG1.raw SUBJECT_SAMPLE_FACTORS - CG2 Genotype:Cgref1-knockout RAW_FILE_NAME=20230329-lipid-CG2.raw SUBJECT_SAMPLE_FACTORS - CG3 Genotype:Cgref1-knockout RAW_FILE_NAME=20230329-lipid-CG3.raw #COLLECTION CO:COLLECTION_SUMMARY Mouse liver tissues were extracted, kept on ice and sent immediately to the the CO:COLLECTION_SUMMARY Centre of Panoromic Sciences (The University of Hong Kong) for testing CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS On ice #TREATMENT TR:TREATMENT_SUMMARY Samples did not receive any treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY As described in the provided protocol (see attached file): - "2mL SP:SAMPLEPREP_SUMMARY chloroform:methanol (2:1, v/v) was added to user provided tissue. The sample was SP:SAMPLEPREP_SUMMARY then sonicated under ice chilled probe sonicator for 20 sec, cooled down for 10 SP:SAMPLEPREP_SUMMARY sec and another sonication for 20 sec. The sample was then centrifuged at 3000 g SP:SAMPLEPREP_SUMMARY for 5 min. Supernatant was aliquot out and dried under nitrogen.The sample was SP:SAMPLEPREP_SUMMARY then reconstituted with IPA:MeOH:chloroform (1:1:0.2, v/v) in 1mg to 5uL. Then, SP:SAMPLEPREP_SUMMARY 3 μL was injected into the LC-MS/MS system." SP:SAMPLEPREP_PROTOCOL_FILENAME untargeted_protocol.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Accucore C30 (150 x 2.1mm,2.6um) CH:SOLVENT_A 10mM ammonium formate with 0.1% formic acid in acetonitrile and water, v/v 6:4 CH:SOLVENT_B 10mM ammonium formate with 0.1% formic acid in acetonitrile and IPA 1:9 CH:FLOW_GRADIENT The gradient started at 30% B and was increased to 43% B in 2 min, then CH:FLOW_GRADIENT increased to 55% B in 2.1 min, 65 % B in 12 min, 85% B in 18 min and 100 % B in CH:FLOW_GRADIENT 20 min, then held for 5min, and decreased linearly to 30% B for re-equilibration CH:FLOW_GRADIENT time at starting conditions. CH:FLOW_RATE 0.26 mL min−1 CH:COLUMN_TEMPERATURE 45°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Orbitrap Exploris 120 Mass Spectrometer MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometry analysis was processed using an Orbitrap Exploris 120 mass MS:MS_COMMENTS spectrometer Thermo Fisher (Waltham, MA, USA) equipped with a HESI II probe in MS:MS_COMMENTS polar switching mode with source parameters set as follows: sheath gas flow MS:MS_COMMENTS rate, 60; auxiliary gas flow rate, 17; sweep gas flow rate, 1; spray MS:MS_COMMENTS voltage,+3.5/-3.0 kV; capillary temperature, 275 oC; S-lens RF level, 70; and MS:MS_COMMENTS heater temperature, 325 °C. Data was collected at dd-MS2 mode. Data analysis MS:MS_COMMENTS was performed using Lipidsearch (Thermofisher Scientific/Mitsui Knowledge MS:MS_COMMENTS Industries) with the default parameters for Orbitrap MS Product Search and MS:MS_COMMENTS Alignment. After alignment, raw peak areas for all identified lipids were MS:MS_COMMENTS exported to Excel files. MS:MS_RESULTS_FILE ST002947_AN004832_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END