#METABOLOMICS WORKBENCH t_ktmt_0211_20240805_184830 DATATRACK_ID:5084 STUDY_ID:ST003452 ANALYSIS_ID:AN005669 PROJECT_ID:PR002126
VERSION             	1
CREATED_ON             	August 28, 2024, 10:43 am
#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
#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
#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
#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           	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           	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           	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
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           	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           	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
#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:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000
CH:COLUMN_NAME                   	Thermo Acclaim (150 x 2.1mm, 3um)
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: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:COLUMN_TEMPERATURE            	50℃
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
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_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Relative abundance (isotope normalized)
MS_METABOLITE_DATA_START
Samples	lipidome_nc_1	lipidome_nc_2	lipidome_nc_3	lipidome_GNB1_1	lipidome_GNB1_2	lipidome_GNB1_3	lipidome_SCARB2_1	lipidome_SCARB2_2	lipidome_SCARB2_3
Factors	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: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:SCARB2	Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2	Sample source:Human subcutaneous adipocytes | Gene Knockdown Target:SCARB2
LPE(22:4)	0.194415916	0.363313336	0.320963586	0.12129017	0.346302454	0.366870128	0.374226514	0.219531662	0.178661389
PE(15:0_24:5)	0.008770126	0.009927218	0.009036028	0.008601428	0.010132129	0.008114132	0.011669713	0.010352754	0.00983851
PE(16:0_20:4)	0.007813013	0.017717606	0.010938387	0.014409328	0.016914212	0.012346197	0.014337357	0.009674035	0.011805803
PE(16:1_18:1)	0.017140389	0.031425842	0.016153226	0.018102799	0.021299004	0.017719976	0.017425542	0.016611072	0.014040322
PE(18:0_20:4)	0.057151692	0.087431483	0.05652753	0.067334518	0.081590767	0.085270261	0.088636957	0.0846679	0.074307716
PE(18:0_22:3)	0.002804163	0.004356677	0.002583149	0.003874243	0.004176938	0.00529542	0.002797783	0.002711419	0.002321213
PE(18:0_22:4)	0.009634258	0.016496918	0.009933761	0.013002772	0.014367558	0.015586713	0.013127548	0.012128969	0.010309382
PE(18:0_22:5)	0.013532661	0.024220438	0.012711772	0.018047061	0.024207924	0.023000721	0.018972747	0.017463143	0.018658099
PE(18:1_20:4)	0.025951675	0.036566061	0.024569796	0.037359525	0.033829469	0.035588023	0.037195991	0.032966349	0.029134581
PE(O-12:1_20:4)	0.010497537	0.012298542	0.010558476	0.012190805	0.011308057	0.012531612	0.012489507	0.009220173	0.009404015
PE(O-16:1_16:1)	0.015129982	0.027406919	0.014682122	0.013195143	0.014790753	0.015325952	0.012370414	0.012007121	0.010399306
PE(O-16:1_18:1)	0.048397347	0.073602929	0.049852802	0.050664337	0.049782397	0.054053594	0.05151929	0.048851518	0.04321655
PE(O-16:1_20:4)	0.107359621	0.151227057	0.105193986	0.12399295	0.118426642	0.130151346	0.150027112	0.150614425	0.125359104
PE(O-16:1_22:6)	0.015786398	0.022391794	0.016045106	0.021022265	0.019735743	0.022133263	0.023131766	0.023800964	0.018517007
PE(O-17:1_20:4)	0.008122028	0.010446016	0.007581222	0.008893676	0.009762725	0.009159274	0.012288474	0.012773639	0.009817601
PE(O-18:1_18:1)	0.015822653	0.022310369	0.015760523	0.019006658	0.018973261	0.020888423	0.020771996	0.020487737	0.017372975
PE(O-18:1_20:3)	0.014346037	0.022892169	0.013853433	0.020383538	0.019612144	0.022036297	0.018507421	0.01889248	0.01492342
PE(O-18:1_20:4)	0.077460493	0.103554939	0.079534224	0.085729565	0.089795279	0.096413549	0.113182576	0.109657962	0.096391113
PE(O-18:1_22:4)	0.022535241	0.033268133	0.015653629	0.027765057	0.026530186	0.031714604	0.031773542	0.028752657	0.019423054
PE(O-18:1_22:6)	0.013831439	0.019729797	0.014444328	0.01764635	0.015464801	0.017826115	0.019150081	0.01433554	0.014446428
PE(O-18:2_18:1)	0.024085645	0.034193827	0.020924894	0.025711789	0.029087459	0.028289327	0.030480188	0.029376588	0.024675744
PE(O-18:2_20:4)	0.093768527	0.123745082	0.094327621	0.097454129	0.102700021	0.112951582	0.133390236	0.118624587	0.104758019
PE(O-18:2_22:4)	0.033782602	0.044747197	0.0291452	0.034989907	0.033446487	0.039310751	0.044249192	0.039496016	0.029195777
PE(O-18:2_22:5)	0.01260647	0.017938681	0.011498616	0.01343783	0.014447114	0.015489746	0.015717872	0.011102162	0.011266595
PE(O-18:2_22:6)	0.012416668	0.018162258	0.011019171	0.015287277	0.014966402	0.016354583	0.016846143	0.0163036	0.012870621
PI(16:0_16:1)	0.019904332	0.036499222	0.017662218	0.024089106	0.022546122	0.021840194	0.016533365	0.017708447	0.01630706
PI(18:0_18:1)	0.067795199	0.079898911	0.063379977	0.087055397	0.086476334	0.097265694	0.079265263	0.078969297	0.075153374
PI(18:0_20:3)	0.124767473	0.161990669	0.09171214	0.195333994	0.189765423	0.239095258	0.107311879	0.096155945	0.10845378
PI(18:0_22:4)	0.029705908	0.034541991	0.021649468	0.043391374	0.040775524	0.04726097	0.034733791	0.032074302	0.029512842
PI(18:0_22:5)	0.01550093	0.020109642	0.011825058	0.032173132	0.026930462	0.038084214	0.024694747	0.021206289	0.019401581
PS(16:0_16:1)	0.010964077	0.02331129	0.011315653	0.012822987	0.013562669	0.010467319	0.010436943	0.009848198	0.009792429
PS(18:0_22:5)	0.011710944	0.016864462	0.010276351	0.013593278	0.009964272	0.014589903	0.012495821	0.010978497	0.007283643
PS(18:0_22:6)	0.008532999	0.011773012	0.008334329	0.012457191	0.014741696	0.011125064	0.012636051	0.012568938	0.010055279
PS(18:1_18:1)	0.023596491	0.028346142	0.019970135	0.028516143	0.033420722	0.032575661	0.023333024	0.024000891	0.018589471
PS(18:1_20:1)	0.036716792	0.056339589	0.032637678	0.059794306	0.053510042	0.048674339	0.055103083	0.042465601	0.039405477
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	BaseRT	MainIon	Calculated m/z
LPE(22:4)	14.9388	M-H	528.3096
PE(15:0_24:5)	34.1454	M-H	778.5392
PE(16:0_20:4)	33.5383	M-H	738.5079
PE(16:1_18:1)	33.418	M-H	714.5079
PE(18:0_20:4)	35.7498	M-H	766.5392
PE(18:0_22:3)	38.4275	M-H	796.5862
PE(18:0_22:4)	37.1489	M-H	794.5705
PE(18:0_22:5)	35.9561	M-H	792.5549
PE(18:1_20:4)	33.878	M-H	764.5236
PE(O-12:1_20:4)	29.7252	M-H	666.4504
PE(O-16:1_16:1)	34.3329	M-H	672.4974
PE(O-16:1_18:1)	36.421	M-H	700.5287
PE(O-16:1_20:4)	35.0192	M-H	722.513
PE(O-16:1_22:6)	34.2741	M-H	746.513
PE(O-17:1_20:4)	35.7555	M-H	736.5287
PE(O-18:1_18:1)	38.328	M-H	728.56
PE(O-18:1_20:3)	37.9283	M-H	752.56
PE(O-18:1_20:4)	36.6578	M-H	750.5443
PE(O-18:1_22:4)	38.1106	M-H	778.5756
PE(O-18:1_22:6)	36.387	M-H	774.5443
PE(O-18:2_18:1)	36.6043	M-H	726.5443
PE(O-18:2_20:4)	34.9384	M-H	748.5287
PE(O-18:2_22:4)	36.6807	M-H	776.56
PE(O-18:2_22:5)	35.1454	M-H	774.5443
PE(O-18:2_22:6)	34.5053	M-H	772.5287
PI(16:0_16:1)	30.3731	M-H	807.5029
PI(18:0_18:1)	34.8275	M-H	863.5655
PI(18:0_20:3)	34.5639	M-H	887.5655
PI(18:0_22:4)	34.7203	M-H	913.5812
PI(18:0_22:5)	33.3241	M-H	911.5655
PS(16:0_16:1)	30.7472	M-H	732.4821
PS(18:0_22:5)	33.8227	M-H	836.5447
PS(18:0_22:6)	33.2294	M-H	834.5291
PS(18:1_18:1)	33.3081	M-H	786.5291
PS(18:1_20:1)	35.3158	M-H	814.5604
METABOLITES_END
#END