#METABOLOMICS WORKBENCH chanpearl_20231026_203513 DATATRACK_ID:4421 STUDY_ID:ST002953 ANALYSIS_ID:AN004850 PROJECT_ID:PR001832 VERSION 1 CREATED_ON November 1, 2023, 6:25 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 Comparisons of hepatic medium & long chain fatty acids expression between wild ST:STUDY_TITLE type and Cgref1-depleted mice of C57BL/6 strain by targeted GC-MS/MS ST:STUDY_SUMMARY In this study, we performed a targeted analysis on the expression of hepatic ST:STUDY_SUMMARY medium & long chain fatty acids between wild type (n=3) and Cgref1-knockout mice ST:STUDY_SUMMARY (n=2) using targeted GC-MS/MS. The results, however, did not reveal significant ST:STUDY_SUMMARY differences between the two groups of mice. In the future, a larger sample size ST:STUDY_SUMMARY and optimization of normalization methods may be helpful to provide more ST:STUDY_SUMMARY meaningful insights. 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=230323_WT1.D SUBJECT_SAMPLE_FACTORS - WT2 Genotype:Wild-type RAW_FILE_NAME=230323_WT2.D SUBJECT_SAMPLE_FACTORS - WT3 Genotype:Wild-type RAW_FILE_NAME=230323_WT3.D SUBJECT_SAMPLE_FACTORS - CG1 Genotype:Cgref1-knockout RAW_FILE_NAME=230323_CG1.D SUBJECT_SAMPLE_FACTORS - CG2 Genotype:Cgref1-knockout RAW_FILE_NAME=230323_CG2.D SUBJECT_SAMPLE_FACTORS - CG3 Genotype:Cgref1-knockout RAW_FILE_NAME=230323_CG3.D #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:COLLECTION_PROTOCOL_FILENAME targeted_protocol.pdf 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): Sample homogenization SP:SAMPLEPREP_SUMMARY and metabolite extraction 100 μl of chloroform with 20 μg C19:0 fatty acid SP:SAMPLEPREP_SUMMARY internal standard was spiked to the sample. The sample was extracted with 5 SP:SAMPLEPREP_SUMMARY rounds of 2:1 Chloroform/Methanol, followed by sonication. After centrifugation, SP:SAMPLEPREP_SUMMARY the supernatant was further cleaned by liquid-liquid extraction in 0.73% NaCl SP:SAMPLEPREP_SUMMARY and Methanol. The resultant mixture was dried under a fume of N2 at 45°C before SP:SAMPLEPREP_SUMMARY transesterification. Transesterification 1 ml of methanol and 50 μl of SP:SAMPLEPREP_SUMMARY concentrated hydrochloric acid (35%, w/w) were added to the sample. The solution SP:SAMPLEPREP_SUMMARY was overlaid with nitrogen and the tube was tightly closed. After vortexing, the SP:SAMPLEPREP_SUMMARY tube was heated at 100°C for 1.5 h. Once cooled to room temperature, 1 ml of SP:SAMPLEPREP_SUMMARY hexane and 1 ml of water were added for FAMEs extraction. The tube was vortexed SP:SAMPLEPREP_SUMMARY and after phase separation, 1 μl the hexane phase was injected for GC-MS SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent DB-23 (60m × 0.25mm, 0.15um) CH:SOLVENT_A N/A CH:SOLVENT_B N/A CH:FLOW_GRADIENT N/A CH:FLOW_RATE N/A CH:COLUMN_TEMPERATURE N/A CH:METHODS_FILENAME targeted_protocol.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE targeted_protocol.pdf #MS MS:INSTRUMENT_NAME Agilent 7010 MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS GC/MS chromatogram was acquired in SCAN and SIM mode in an Agilent 7890B GC - MS:MS_COMMENTS Agilent 7010 Triple Quadrapole Mass Spectrometer system. The sample was MS:MS_COMMENTS separated through an Agilent DB-23 capillary column (60 m × 0.25 mm ID, 0.15 MS:MS_COMMENTS μm film thickness) under constant pressure of helium at 33.4 psi. The GC oven MS:MS_COMMENTS program started at 50°C (hold time 1 min) and was increased to 175°C at a ramp MS:MS_COMMENTS rate of 25°C/min. The temperature was then raised to 190°C (hold time 5 min) MS:MS_COMMENTS at a ramp rate of 3.5°C/min. Finally the temperature was raised to 220°C (hold MS:MS_COMMENTS time 4 min) at a ramp rate of 2°C/min. Inlet temperature and transfer line MS:MS_COMMENTS temperature were 250°C and 280°C respectively. Characteristic fragment ions MS:MS_COMMENTS (m/z 55, 67, 69, 74, 79, 81, 83, 87, 91, 93, 95, 96, 97, 115, 127, 143) were MS:MS_COMMENTS monitored in SIM mode throughout the run. Mass spectra from m/z 50-350 were MS:MS_COMMENTS acquired in SCAN mode. Data analysis was performed using the Agilent MassHunter MS:MS_COMMENTS Workstation Quantitative Analysis Software. Linear calibration curves for each MS:MS_COMMENTS analyte were generated by plotting peak area ratio of external/internal standard MS:MS_COMMENTS against standard concentration at different concentration level. Analytes were MS:MS_COMMENTS confirmed by comparing the ratio of characteristic fragment ions in the sample MS:MS_COMMENTS and standard. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/g MS_METABOLITE_DATA_START Samples WT1 WT2 WT3 CG1 CG2 CG3 Factors Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Cgref1-knockout Genotype:Cgref1-knockout Genotype:Cgref1-knockout Butyric Acid 0.00 0.00 0.00 0.00 0.00 0.00 Caproic Acid 3.07 0.00 0.00 0.00 0.00 0.00 Caprylic Acid 8.48 0.00 0.00 0.00 0.00 0.00 Capric Acid 8.01 0.00 0.00 3.49 0.00 3.50 Undecanoic Acid 0.00 0.00 0.00 0.00 0.00 0.00 Lauric Acid 36.08 0.00 0.00 11.65 0.00 18.99 Tridecanoic Acid 0.99 0.00 0.00 0.00 0.00 1.45 Myristic Acid 116.02 134.19 102.16 110.42 61.22 224.10 Myristoleic Acid 5.92 5.50 4.51 3.04 1.61 12.11 Pentadecanoic Acid 48.39 53.39 39.13 53.09 41.67 88.10 cis-10-Pentadecenoic Acid 0.00 0.00 0.00 0.00 0.00 0.00 Palmitic Acid 4624.96 5261.28 5030.93 5212.14 4136.63 5065.24 Palmitoleic Acid 1342.91 1571.11 1266.01 890.94 479.89 1665.68 Heptadecanoic Acid 136.17 150.63 112.84 167.23 154.09 247.58 cis-10-Heptadecenoic Acid 0.00 0.00 0.00 0.00 0.00 0.00 Stearic Acid 3035.67 3906.11 3532.51 4022.06 3460.67 3497.52 Elaidic Acid 0.00 0.00 0.00 0.00 0.00 0.00 Oleic Acid 3731.43 4133.67 3885.79 3684.61 2609.93 4208.96 Linolelaidic Acid 0.00 0.00 0.00 0.00 0.00 0.00 Linoleic Acid 10080.84 11204.31 10312.39 11877.94 9004.47 12278.38 γ-Linolenic Acid 207.35 264.29 168.28 406.85 295.54 579.78 α-Linolenic Acid 1104.89 1675.42 1060.14 1683.82 706.39 3334.66 Arachidic Acid 50.15 119.30 89.79 129.55 124.08 55.19 cis-11- Eicosenoic Acid 131.95 139.22 131.24 118.80 92.92 168.43 cis-11,14- Eicosadienoic Acid 216.11 166.76 174.04 156.90 148.93 305.00 Henicosanoic Acid 0.00 0.00 0.00 0.00 0.00 0.00 cis-8,11,14-Eicosatrienoic Acid 1635.53 2081.75 1900.69 1055.79 967.36 1816.46 Arachidonic Acid 20824.09 23486.92 22006.89 22561.23 20661.06 22992.47 cis-11,14,17-Eicosatrienoic Acid 33.55 35.40 33.62 34.45 28.52 43.99 cis-5,8,11,14,17-Eicosapentaenoic Acid 2667.76 4895.54 2674.58 4065.88 2462.81 4870.97 Behenic Acid 41.98 115.15 108.92 134.18 156.68 43.61 Erucic Acid 15.31 14.51 13.29 13.39 10.67 10.21 cis-13,16-Docosadienoic Acid 5.99 5.88 6.40 6.34 5.73 6.45 Tricosanoic Acid 13.68 14.79 14.08 17.74 17.59 18.21 Lignoceric Acid 38.27 58.71 53.94 75.82 71.35 44.27 cis-4,7,10,13,16,19-Docosahexaenoic Acid 43586.14 54490.34 43517.18 53720.96 45320.63 52233.35 Nervonic Acid 61.21 52.39 60.96 56.05 53.13 76.62 Hexacosanoic Acid 0.00 0.00 0.00 0.00 0.00 0.00 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Butyric Acid Caproic Acid Caprylic Acid Capric Acid Undecanoic Acid Lauric Acid Tridecanoic Acid Myristic Acid Myristoleic Acid Pentadecanoic Acid cis-10-Pentadecenoic Acid Palmitic Acid Palmitoleic Acid Heptadecanoic Acid cis-10-Heptadecenoic Acid Stearic Acid Elaidic Acid Oleic Acid Linolelaidic Acid Linoleic Acid γ-Linolenic Acid α-Linolenic Acid Arachidic Acid cis-11- Eicosenoic Acid cis-11,14- Eicosadienoic Acid Henicosanoic Acid cis-8,11,14-Eicosatrienoic Acid Arachidonic Acid cis-11,14,17-Eicosatrienoic Acid cis-5,8,11,14,17-Eicosapentaenoic Acid Behenic Acid Erucic Acid cis-13,16-Docosadienoic Acid Tricosanoic Acid Lignoceric Acid cis-4,7,10,13,16,19-Docosahexaenoic Acid Nervonic Acid Hexacosanoic Acid METABOLITES_END #END