#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 × 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            	50℃
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