#METABOLOMICS WORKBENCH hienbui_20250306_073923 DATATRACK_ID:5700 STUDY_ID:ST003808 ANALYSIS_ID:AN006262 PROJECT_ID:PR002382
VERSION             	1
CREATED_ON             	March 21, 2025, 11:35 am
#PROJECT
PR:PROJECT_TITLE                 	Lipidomics of human mammary epithelial stem-like cells (hMECs) overexpressing
PR:PROJECT_TITLE                 	Glucose-6-phosphate dehydrogenase (G6PD)
PR:PROJECT_SUMMARY               	Peroxisomes play a crucial role in cellular metabolism. Glucose-6-phosphate
PR:PROJECT_SUMMARY               	dehydrogenase (G6PD), the gatekeeper enzyme of the pentose phosphate pathway, is
PR:PROJECT_SUMMARY               	primarily localized in the cytosol. However, studies have reported its presence
PR:PROJECT_SUMMARY               	in peroxisomes as well. This project aims to determine the function of G6PD on
PR:PROJECT_SUMMARY               	the peroxisomal membrane. To investigate this, we overexpressed G6PD either in
PR:PROJECT_SUMMARY               	the cytosol or on the peroxisomal membrane of human mammary epithelial stem-like
PR:PROJECT_SUMMARY               	cells (hMECs), a cell line that retains characteristics of primary mammary
PR:PROJECT_SUMMARY               	epithelial stem cells, and analyzed their lipidomic profiles for comparison.
PR:INSTITUTE                     	University of Helsinki
PR:DEPARTMENT                    	Faculty of Biological and Environmental Sciences
PR:LABORATORY                    	Katajisto Laboratory
PR:LAST_NAME                     	Bui
PR:FIRST_NAME                    	Hien
PR:ADDRESS                       	Biocenter 1, Viikinkaari 9, Helsinki, Uusimaa, 00790, Finland
PR:EMAIL                         	hien.bui@helsinki.fi
PR:PHONE                         	+358294159407
#STUDY
ST:STUDY_TITLE                   	Glucose-6-phosphate-dehydrogenase on old peroxisomes maintains self-renewal of
ST:STUDY_TITLE                   	epithelial stem cells after asymmetric cell division
ST:STUDY_SUMMARY                 	Peroxisomes play a crucial role in cellular metabolism. Glucose-6-phosphate
ST:STUDY_SUMMARY                 	dehydrogenase (G6PD), the gatekeeper enzyme of the pentose phosphate pathway, is
ST:STUDY_SUMMARY                 	primarily localized in the cytosol. However, studies have reported its presence
ST:STUDY_SUMMARY                 	in peroxisomes as well. This project aims to determine the function of G6PD on
ST:STUDY_SUMMARY                 	the peroxisomal membrane. In this study, we overexpressed G6PD in either the
ST:STUDY_SUMMARY                 	cytosol or on the peroxisomal membrane of mammary epithelial stem-like cells
ST:STUDY_SUMMARY                 	(hMECs). By comparing their lipidomic profiles, we found that peroxisomal
ST:STUDY_SUMMARY                 	membrane-associated G6PD provides NADPH, which feeds into peroxisomal ether
ST:STUDY_SUMMARY                 	lipid synthesis.
ST:INSTITUTE                     	University of Helsinki
ST:DEPARTMENT                    	Faculty of Biological and Environmental Sciences
ST:LABORATORY                    	Katajisto Laboratory
ST:LAST_NAME                     	Hien
ST:FIRST_NAME                    	Bui
ST:ADDRESS                       	Biocenter 1, Viikinkaari 9
ST:EMAIL                         	hien.bui@helsinki.fi
ST:PHONE                         	+358294159407
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	-	c-pos-1	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Overexpressed	Replicate=rep1; RAW_FILE_NAME(Raw_file_name)=c-pos-1_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	c-neg-2	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Non-overexpressed	Replicate=rep1; RAW_FILE_NAME(Raw_file_name)=c-neg-2_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	p-pos-3	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Overexpressed	Replicate=rep1; RAW_FILE_NAME(Raw_file_name)=p-pos-3_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	p-neg-4	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Non-overexpressed	Replicate=rep1; RAW_FILE_NAME(Raw_file_name)=p-neg-4_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	c-pos-5	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Overexpressed	Replicate=rep2; RAW_FILE_NAME(Raw_file_name)=c-pos-5_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	c-neg-6	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Non-overexpressed	Replicate=rep2; RAW_FILE_NAME(Raw_file_name)=c-neg-6_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	p-pos-7	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Overexpressed	Replicate=rep2; RAW_FILE_NAME(Raw_file_name)=p-pos-7_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	p-neg-8	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Non-overexpressed	Replicate=rep2; RAW_FILE_NAME(Raw_file_name)=p-neg-8_07032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	c-pos-9	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Overexpressed	Replicate=rep3; RAW_FILE_NAME(Raw_file_name)=c-pos-9_07032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	c-neg-10	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Non-overexpressed	Replicate=rep3; RAW_FILE_NAME(Raw_file_name)=c-neg-10_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	p-pos-11	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Overexpressed	Replicate=rep3; RAW_FILE_NAME(Raw_file_name)=p-pos-11_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	p-neg-12	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Non-overexpressed	Replicate=rep3; RAW_FILE_NAME(Raw_file_name)=p-neg-12_07032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	c-pos-13	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Overexpressed	Replicate=rep4; RAW_FILE_NAME(Raw_file_name)=c-pos-13_07032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	c-neg-14	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Non-overexpressed	Replicate=rep4; RAW_FILE_NAME(Raw_file_name)=c-neg-14_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	p-pos-15	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Overexpressed	Replicate=rep4; RAW_FILE_NAME(Raw_file_name)=p-pos-15_06032024-1.CDF
SUBJECT_SAMPLE_FACTORS           	-	p-neg-16	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Non-overexpressed	Replicate=rep4; RAW_FILE_NAME(Raw_file_name)=p-neg-16_07032024-1.CDF
#COLLECTION
CO:COLLECTION_SUMMARY            	Human mammary epithelial cells (hMECs) line FL2 (PMID: 21498687) was used for
CO:COLLECTION_SUMMARY            	this study. hMECs were maintained in MEGM mammary epithelial medium (Lonza,
CO:COLLECTION_SUMMARY            	CC-3153). G6PD overexpression plasmids were generated in a pRP backbone by
CO:COLLECTION_SUMMARY            	inserting the G6PD sequence with or without peroxisomal targeting signal
CO:COLLECTION_SUMMARY            	downstream of the hPGK promoter. In addition, the plasmids contain an mCherry
CO:COLLECTION_SUMMARY            	sequence downstream of an IRES sequence. mCherry was used as the selection
CO:COLLECTION_SUMMARY            	marker for transfected cells in FACS sorting. To over-express G6PD in hMECs,
CO:COLLECTION_SUMMARY            	endotoxin-free plasmids were transfected to the cells using jetPRIME (Polyplus,
CO:COLLECTION_SUMMARY            	101000015). 24hours post-transfection, cells were trypsinized, sorted into
CO:COLLECTION_SUMMARY            	culture media by Fluorescence-Activated Cell Sorting (FACS), centrifuged at 500g
CO:COLLECTION_SUMMARY            	for 5min, washed 1 with cold PBS following by washed 1 with sucrose 0.25M.
CO:COLLECTION_SUMMARY            	Pellets were snap freeze with liquid N2 and store at -70.
CO:SAMPLE_TYPE                   	Epithelial cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Human mammary epithelial stem like cells (hMECs) were transfected with
TR:TREATMENT_SUMMARY             	endotoxin-free G6PD overexpression plasmids using jetPRIME (Polyplus,
TR:TREATMENT_SUMMARY             	101000015). Cells were analyzed 24h post-transfection.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	GC-MS: Fatty acyl (FA) and alkenyl chains, and fatty alcohols (FOHs) were
SP:SAMPLEPREP_SUMMARY            	analysed by gas chromatography (GC). Aliquots of cell pellet Folch-extracts were
SP:SAMPLEPREP_SUMMARY            	evaporated near to dryness under nitrogen flow and the extracted lipids were
SP:SAMPLEPREP_SUMMARY            	transmethylated by heating with 1% H2SO4 in methanol under nitrogen atmosphere.
SP:SAMPLEPREP_SUMMARY            	This converts FAs to their methyl esters (FAMEs), phospholipid-derived alkenyl
SP:SAMPLEPREP_SUMMARY            	chains to dimethyl acetals (DMAs) and leaves FOHs (1-ol) intact. After adding
SP:SAMPLEPREP_SUMMARY            	water, the FAMEs, DMAs and FOHs were extracted with hexane, and the sample
SP:SAMPLEPREP_SUMMARY            	solution was dried, and concentrated. LC-MS: Lipids were extracted from cell
SP:SAMPLEPREP_SUMMARY            	pellets (at least from one million cells) according to Folch et al (PMID:
SP:SAMPLEPREP_SUMMARY            	13428781), and dissolved in chloroform/methanol 1:2 (v/v). Internal standards
SP:SAMPLEPREP_SUMMARY            	[EquiSPLASH® internal standard mixture and Ceramide (Cer) 18:1;O2/17:0, both
SP:SAMPLEPREP_SUMMARY            	from Merck] were added and samples were analyzed with LC-MS/MS.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	The method used acetonitrile/water/isopropanol-based solvent system, Agilent
CH:CHROMATOGRAPHY_SUMMARY        	1290 Infinity HPLC (Agilent Technologies, Santa Clara, CA) equipped with a Luna
CH:CHROMATOGRAPHY_SUMMARY        	Omega C18 100 Å (50 x 2.1 mm, 1.6 μm) column (Phenomenex) and Agilent 6490
CH:CHROMATOGRAPHY_SUMMARY        	Triple Quad LC/MS with iFunnel Technology. The lipids species were identified
CH:CHROMATOGRAPHY_SUMMARY        	and quantified using lipid class-specific precursor ion and neutral loss
CH:CHROMATOGRAPHY_SUMMARY        	detection modes. PE plasmalogens (PE P) were identified with alkenyl chain
CH:CHROMATOGRAPHY_SUMMARY        	(16:0, 18:0, 18:1)-specific scans and quantified from MS- scan. - Due to a
CH:CHROMATOGRAPHY_SUMMARY        	contamination during the sample run, some samples were removed from analysis
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity
CH:COLUMN_NAME                   	Phenomenex Luna Omega C18 (50 x 2.1mm, 1.6um, 100 Å)
CH:SOLVENT_A                     	40% water/60% acetonitrile; 0.1% formic acid; 10mM ammonium formate
CH:SOLVENT_B                     	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10mM ammonium formate
CH:FLOW_GRADIENT                 	32% B at 0.0 min, 32% B at 1.5 min, 45% B at 4.0 min, 52% B at 5.0 min, 58% B at
CH:FLOW_GRADIENT                 	8.0 min, 66% B at 11.0 min, 70% B at 14.0 min, 75% B at 18.0 min, 97% B at 21.0
CH:FLOW_GRADIENT                 	min, 97% B at 25.0 min, 32% B at 26.0 min, 32% B at 32.0 min. flow gradient
CH:FLOW_GRADIENT                 	field is a linear gradient
CH:FLOW_RATE                     	0.200 mL/min
CH:COLUMN_TEMPERATURE            	25
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6490 QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	PE plasmalogens (PE P) were identified with alkenyl chain (16:0, 18:0,
MS:MS_COMMENTS                   	18:1)-specific scans and quantified from MS- scan. Mass spectra were processed
MS:MS_COMMENTS                   	using MassHunter Qualitative Navigator software (Agilent) and lipid species were
MS:MS_COMMENTS                   	quantified utilizing the internal and additional external standards
MS:MS_COMMENTS                   	(glucosylceramide 18:1;O2/24:1) and LIMSA software. Lipid data are expressed as
MS:MS_COMMENTS                   	molar percentages (mol%) and lipid species are marked as follows: [sum of acyl
MS:MS_COMMENTS                   	chain carbons]: [sum of acyl chain double bonds] (e.g., PC 36:1).
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	pmol/sample
MS_METABOLITE_DATA_START
Samples	c-pos-1	c-neg-2	p-neg-4	c-pos-5	c-neg-6	p-pos-7	c-neg-10	p-pos-11	p-neg-12	c-pos-13	p-pos-15	p-neg-16
Factors	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Non-overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Non-overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Non-overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Non-overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Non-overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:cytosolic G6PD | G6PD expression:Overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Overexpressed	Sample source:human mammary epithelial stem-like cell | Sample type:peroxisomal G6PD | G6PD expression:Non-overexpressed
PE P-32:1	290.604	223.879	205.002	264.803	249.442	230.649	317.833	366.788	288.642	348.060	393.611	355.247
PE P-34:1	1501.714	937.731	1033.682	1398.168	1243.596	1291.715	1518.294	1942.099	1481.794	2358.030	2210.815	1791.309
PE P-34:2	210.028	164.668	159.835	204.098	233.820	192.971	246.388	290.668	251.717	282.978	323.249	260.306
PE P-36:1	823.642	486.487	438.520	787.058	690.444	772.168	678.917	1189.194	817.286	1103.012	1378.104	908.408
PE P-36:2	657.944	382.271	416.513	545.020	518.587	574.278	661.412	857.139	575.425	897.811	923.041	717.410
PE P-36:4	87.811	55.559	43.043	96.249	77.992	76.563	62.489	120.892	73.649	144.297	155.874	125.574
PE P-38:4	94.326	51.569	49.254	77.143	80.872	76.846	73.772	149.561	97.657	134.418	145.411	113.657
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	m/z
PE P-32:1	672.5
PE P-34:1	700.5
PE P-34:2	698.5
PE P-36:1	728.6
PE P-36:2	726.5
PE P-36:4	722.5
PE P-38:4	750.5
METABOLITES_END
#END