#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