#METABOLOMICS WORKBENCH t_ktmt_0211_20240805_184830 DATATRACK_ID:5084 STUDY_ID:ST003452 ANALYSIS_ID:AN005669 PROJECT_ID:PR002126 VERSION 1 CREATED_ON 03-30-2025 #PROJECT PR:PROJECT_TITLE Integrative proteomic and lipidomic analysis of GNB1 and SCARB2 knockdown in PR:PROJECT_TITLE human subcutaneous adipocytes PR:PROJECT_TYPE Multi-omics (Proteomics and Lipidomics) PR:PROJECT_SUMMARY This project focuses on the knockdown of two candidate genes, GNB1 and SCARB2, PR:PROJECT_SUMMARY identified through a comprehensive integration of genome-wide association PR:PROJECT_SUMMARY studies (GWAS) related to BMI and proteomics data from previous studies using PR:PROJECT_SUMMARY human subcutaneous adipocytes before and after fat accumulation. We aim to PR:PROJECT_SUMMARY understand the regulatory roles of these genes in fat accumulation by performing PR:PROJECT_SUMMARY an integrated analysis of changes in the proteome and lipidome using mass PR:PROJECT_SUMMARY spectrometry following gene knockdown. PR:INSTITUTE Hamamatsu University School of Medicine PR:LAST_NAME Kitamoto PR:FIRST_NAME Takuya PR:ADDRESS 1-20-1 Handayama, Chuo-ku, Hamamatsu 431-3192, Japan PR:EMAIL t.ktmt@hama-med.ac.jp PR:PHONE +81-53-435-2987 PR:PUBLICATIONS DOI: 10.1371/journal.pone.0319163 PR:DOI http://dx.doi.org/10.21228/M8T52T #STUDY ST:STUDY_TITLE Integrated Proteomic and Lipidomic Analysis ST:STUDY_SUMMARY GNB1 and SCARB2 genes were knocked down in human subcutaneous adipocytes using ST:STUDY_SUMMARY RNA interference. Both proteomic and lipidomic analyses were performed using ST:STUDY_SUMMARY mass spectrometry. The proteomic results revealed significant changes in the ST:STUDY_SUMMARY expression levels of key proteins involved in lipid metabolism and adipogenesis. ST:STUDY_SUMMARY The lipidomic analysis identified significant changes in lipid species, ST:STUDY_SUMMARY including phosphatidylcholines, ceramides, and cholesterol esters. This ST:STUDY_SUMMARY integrated analysis provides insights into the molecular pathways and lipid ST:STUDY_SUMMARY metabolism regulated by GNB1 and SCARB2 in adipocyte function. ST:INSTITUTE Hamamatsu University School of Medicine ST:LAST_NAME Kitamoto ST:FIRST_NAME Takuya ST:ADDRESS 1-20-1 Handayama, Chuo-ku, Hamamatsu 431-3192, Japan ST:EMAIL t.ktmt@hama-med.ac.jp ST:PHONE +81-53-435-2987 ST:SUBMIT_DATE 2024-08-05 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 38 SU:GENDER Female SU:CELL_BIOSOURCE_OR_SUPPLIER Zen-Bio, Inc. (Research Triangle Park, NC, USA) SU:SUBJECT_COMMENTS The cell line was derived from abdominal subcutaneous adipose tissue SU:SPECIES_GROUP Mammals #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS GNB1_1 lipidome_GNB1_1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Analysis Method=lipidome; RAW_FILE_NAME=lipidome_GNB1_1.raw SUBJECT_SAMPLE_FACTORS GNB1_2 lipidome_GNB1_2 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Analysis Method=lipidome; RAW_FILE_NAME=lipidome_GNB1_2.raw SUBJECT_SAMPLE_FACTORS GNB1_3 lipidome_GNB1_3 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Analysis Method=lipidome; RAW_FILE_NAME=lipidome_GNB1_3.raw SUBJECT_SAMPLE_FACTORS GNB1_1 proteome_GNB1_1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Analysis Method=proteome; RAW_FILE_NAME=proteome_GNB1_1.raw SUBJECT_SAMPLE_FACTORS GNB1_2 proteome_GNB1_2 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Analysis Method=proteome; RAW_FILE_NAME=proteome_GNB1_2.raw SUBJECT_SAMPLE_FACTORS GNB1_3 proteome_GNB1_3 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Analysis Method=proteome; RAW_FILE_NAME=proteome_GNB1_3.raw SUBJECT_SAMPLE_FACTORS NC_1 lipidome_nc_1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Analysis Method=lipidome; RAW_FILE_NAME=lipidome_nc_1.raw SUBJECT_SAMPLE_FACTORS NC_2 lipidome_nc_2 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Analysis Method=lipidome; RAW_FILE_NAME=lipidome_nc_2.raw SUBJECT_SAMPLE_FACTORS NC_3 lipidome_nc_3 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Analysis Method=lipidome; RAW_FILE_NAME=lipidome_nc_3.raw SUBJECT_SAMPLE_FACTORS NC_1 proteome_nc_1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Analysis Method=proteome; RAW_FILE_NAME=proteome_nc_1.raw SUBJECT_SAMPLE_FACTORS NC_2 proteome_nc_2 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Analysis Method=proteome; RAW_FILE_NAME=proteome_nc_2.raw SUBJECT_SAMPLE_FACTORS NC_3 proteome_nc_3 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Analysis Method=proteome; RAW_FILE_NAME=proteome_nc_3.raw SUBJECT_SAMPLE_FACTORS SCARB2_1 lipidome_SCARB2_1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 Analysis Method=lipidome; RAW_FILE_NAME=lipidome_SCARB2_1.raw SUBJECT_SAMPLE_FACTORS SCARB2_2 lipidome_SCARB2_2 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 Analysis Method=lipidome; RAW_FILE_NAME=lipidome_SCARB2_2.raw SUBJECT_SAMPLE_FACTORS SCARB2_3 lipidome_SCARB2_3 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 Analysis Method=lipidome; RAW_FILE_NAME=lipidome_SCARB2_3.raw SUBJECT_SAMPLE_FACTORS SCARB2_1 proteome_SCARB2_1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 Analysis Method=proteome; RAW_FILE_NAME=proteome_SCARB2_1.raw SUBJECT_SAMPLE_FACTORS SCARB2_2 proteome_SCARB2_2 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 Analysis Method=proteome; RAW_FILE_NAME=proteome_SCARB2_2.raw SUBJECT_SAMPLE_FACTORS SCARB2_3 proteome_SCARB2_3 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 Analysis Method=proteome; RAW_FILE_NAME=proteome_SCARB2_3.raw #COLLECTION CO:COLLECTION_SUMMARY Primary cultured human preadipocytes were obtained from Zen-Bio, Inc., derived CO:COLLECTION_SUMMARY from the abdominal subcutaneous adipose tissue of a 38-year-old Caucasian woman. CO:COLLECTION_SUMMARY The cells were grown, differentiated, and collected as mature adipocytes on day CO:COLLECTION_SUMMARY 14. All experiments were performed in three independent replicates. CO:SAMPLE_TYPE White adipose #TREATMENT TR:TREATMENT_SUMMARY siRNA transfection was performed to knock down the expression of GNB1 and SCARB2 TR:TREATMENT_SUMMARY genes in differentiated human subcutaneous adipocytes. Three rounds of TR:TREATMENT_SUMMARY transfection were carried out over a period of 14 days. Following the final TR:TREATMENT_SUMMARY transfection, cells were harvested for both proteomic and lipidomic analyses. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Proteomics: Cells were lysed, proteins were extracted, and concentrations were SP:SAMPLEPREP_SUMMARY determined using the BCA assay. Proteins were precipitated, reduced, alkylated, SP:SAMPLEPREP_SUMMARY and digested with trypsin. Peptides were purified using a MonoSpin C18 column SP:SAMPLEPREP_SUMMARY and analyzed using an EASY-nLC 1200 and a Q-Exactive Orbitrap mass spectrometer SP:SAMPLEPREP_SUMMARY (Thermo Fisher Scientific). Data were processed using Proteome Discoverer 2.2 SP:SAMPLEPREP_SUMMARY software (Thermo Fisher Scientific). Lipidomics: Lipids were extracted using the SP:SAMPLEPREP_SUMMARY Bligh and Dyer method, dissolved in methanol, and analyzed using an Ultimate SP:SAMPLEPREP_SUMMARY 3000 and a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). SP:SAMPLEPREP_SUMMARY Data were processed using LipidSearch software version 5.1.9 (Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY HPLC lipidome analysis. C18 column (Acclaim 120 C18 column (3 μm, 2.1 mm à 150 CH:CHROMATOGRAPHY_SUMMARY mm), gradient elution with aqueous and organic solvents containing ammonium CH:CHROMATOGRAPHY_SUMMARY formate and formic acid. Flow rate: 300 μL/min, column at 50°C. 70-min CH:CHROMATOGRAPHY_SUMMARY gradient. Solvent A: 5 mM ammonium formate in water/methanol/acetonitrile CH:CHROMATOGRAPHY_SUMMARY (2:1:1) with 0.1% formic acid; Solvent B: 5 mM ammonium formate in CH:CHROMATOGRAPHY_SUMMARY isopropanol/acetonitrile (9:1) with 0.1% formic acid CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Thermo Acclaim (150 x 2.1mm, 3um) CH:COLUMN_TEMPERATURE 50â CH:FLOW_GRADIENT 20% to 100% B over 50 min, 100% B for 10 min, then return to 20% B CH:FLOW_RATE 300 μL/min CH:SOLVENT_A 50% water/25% methanol/25% acetonitrile; 5 mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 5 mM ammonium formate; 0.1% formic acid CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS MS acquisition Comments: Full scan MS followed by data-dependent MS/MS Data MS:MS_COMMENTS processing Comments: Raw data processed using LipidSearch for lipidomics MS:MS_COMMENTS Software/procedures used for feature assignments: LipidSearch for lipid MS:MS_COMMENTS identification MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Relative abundance (isotope normalized) MS_METABOLITE_DATA_START Samples lipidome_GNB1_1 lipidome_GNB1_2 lipidome_GNB1_3 lipidome_nc_1 lipidome_nc_2 lipidome_nc_3 lipidome_SCARB2_1 lipidome_SCARB2_2 lipidome_SCARB2_3 Factors Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:GNB1 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:Negative Control Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2 LPE(22:4) 0.1213 0.3463 0.3669 0.1944 0.3633 0.3210 0.3742 0.2195 0.1787 PE(15:0_24:5) 0.0086 0.0101 0.0081 0.0088 0.0099 0.0090 0.0117 0.0104 0.0098 PE(16:0_20:4) 0.0144 0.0169 0.0123 0.0078 0.0177 0.0109 0.0143 0.0097 0.0118 PE(16:1_18:1) 0.0181 0.0213 0.0177 0.0171 0.0314 0.0162 0.0174 0.0166 0.0140 PE(18:0_20:4) 0.0673 0.0816 0.0853 0.0572 0.0874 0.0565 0.0886 0.0847 0.0743 PE(18:0_22:3) 0.0039 0.0042 0.0053 0.0028 0.0044 0.0026 0.0028 0.0027 0.0023 PE(18:0_22:4) 0.0130 0.0144 0.0156 0.0096 0.0165 0.0099 0.0131 0.0121 0.0103 PE(18:0_22:5) 0.0180 0.0242 0.0230 0.0135 0.0242 0.0127 0.0190 0.0175 0.0187 PE(18:1_20:4) 0.0374 0.0338 0.0356 0.0260 0.0366 0.0246 0.0372 0.0330 0.0291 PE(O-12:1_20:4) 0.0122 0.0113 0.0125 0.0105 0.0123 0.0106 0.0125 0.0092 0.0094 PE(O-16:1_16:1) 0.0132 0.0148 0.0153 0.0151 0.0274 0.0147 0.0124 0.0120 0.0104 PE(O-16:1_18:1) 0.0507 0.0498 0.0541 0.0484 0.0736 0.0499 0.0515 0.0489 0.0432 PE(O-16:1_20:4) 0.1240 0.1184 0.1302 0.1074 0.1512 0.1052 0.1500 0.1506 0.1254 PE(O-16:1_22:6) 0.0210 0.0197 0.0221 0.0158 0.0224 0.0160 0.0231 0.0238 0.0185 PE(O-17:1_20:4) 0.0089 0.0098 0.0092 0.0081 0.0104 0.0076 0.0123 0.0128 0.0098 PE(O-18:1_18:1) 0.0190 0.0190 0.0209 0.0158 0.0223 0.0158 0.0208 0.0205 0.0174 PE(O-18:1_20:3) 0.0204 0.0196 0.0220 0.0143 0.0229 0.0139 0.0185 0.0189 0.0149 PE(O-18:1_20:4) 0.0857 0.0898 0.0964 0.0775 0.1036 0.0795 0.1132 0.1097 0.0964 PE(O-18:1_22:4) 0.0278 0.0265 0.0317 0.0225 0.0333 0.0157 0.0318 0.0288 0.0194 PE(O-18:1_22:6) 0.0176 0.0155 0.0178 0.0138 0.0197 0.0144 0.0192 0.0143 0.0144 PE(O-18:2_18:1) 0.0257 0.0291 0.0283 0.0241 0.0342 0.0209 0.0305 0.0294 0.0247 PE(O-18:2_20:4) 0.0975 0.1027 0.1130 0.0938 0.1237 0.0943 0.1334 0.1186 0.1048 PE(O-18:2_22:4) 0.0350 0.0334 0.0393 0.0338 0.0447 0.0291 0.0442 0.0395 0.0292 PE(O-18:2_22:5) 0.0134 0.0144 0.0155 0.0126 0.0179 0.0115 0.0157 0.0111 0.0113 PE(O-18:2_22:6) 0.0153 0.0150 0.0164 0.0124 0.0182 0.0110 0.0168 0.0163 0.0129 PI(16:0_16:1) 0.0241 0.0225 0.0218 0.0199 0.0365 0.0177 0.0165 0.0177 0.0163 PI(18:0_18:1) 0.0871 0.0865 0.0973 0.0678 0.0799 0.0634 0.0793 0.0790 0.0752 PI(18:0_20:3) 0.1953 0.1898 0.2391 0.1248 0.1620 0.0917 0.1073 0.0962 0.1085 PI(18:0_22:4) 0.0434 0.0408 0.0473 0.0297 0.0345 0.0216 0.0347 0.0321 0.0295 PI(18:0_22:5) 0.0322 0.0269 0.0381 0.0155 0.0201 0.0118 0.0247 0.0212 0.0194 PS(16:0_16:1) 0.0128 0.0136 0.0105 0.0110 0.0233 0.0113 0.0104 0.0098 0.0098 PS(18:0_22:5) 0.0136 0.0100 0.0146 0.0117 0.0169 0.0103 0.0125 0.0110 0.0073 PS(18:0_22:6) 0.0125 0.0147 0.0111 0.0085 0.0118 0.0083 0.0126 0.0126 0.0101 PS(18:1_18:1) 0.0285 0.0334 0.0326 0.0236 0.0283 0.0200 0.0233 0.0240 0.0186 PS(18:1_20:1) 0.0598 0.0535 0.0487 0.0367 0.0563 0.0326 0.0551 0.0425 0.0394 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant LPE(22:4) 528.3096 PE(15:0_24:5) 778.5392 PE(16:0_20:4) 738.5079 PE(16:1_18:1) 714.5079 PE(18:0_20:4) 766.5392 PE(18:0_22:3) 796.5862 PE(18:0_22:4) 794.5705 PE(18:0_22:5) 792.5549 PE(18:1_20:4) 764.5236 PE(O-12:1_20:4) 666.4504 PE(O-16:1_16:1) 672.4974 PE(O-16:1_18:1) 700.5287 PE(O-16:1_20:4) 722.513 PE(O-16:1_22:6) 746.513 PE(O-17:1_20:4) 736.5287 PE(O-18:1_18:1) 728.56 PE(O-18:1_20:3) 752.56 PE(O-18:1_20:4) 750.5443 PE(O-18:1_22:4) 778.5756 PE(O-18:1_22:6) 774.5443 PE(O-18:2_18:1) 726.5443 PE(O-18:2_20:4) 748.5287 PE(O-18:2_22:4) 776.56 PE(O-18:2_22:5) 774.5443 PE(O-18:2_22:6) 772.5287 PI(16:0_16:1) 807.5029 PI(18:0_18:1) 863.5655 PI(18:0_20:3) 887.5655 PI(18:0_22:4) 913.5812 PI(18:0_22:5) 911.5655 PS(16:0_16:1) 732.4821 PS(18:0_22:5) 836.5447 PS(18:0_22:6) 834.5291 PS(18:1_18:1) 786.5291 PS(18:1_20:1) 814.5604 METABOLITES_END #END