#METABOLOMICS WORKBENCH parthosen_20210131_164908 DATATRACK_ID:2439 STUDY_ID:ST001678 ANALYSIS_ID:AN002736 PROJECT_ID:000000 VERSION 1 CREATED_ON February 3, 2021, 9:28 am #PROJECT PR:PROJECT_TITLE Quantitative analysis and genome-scale modeling of human CD4+ T-cell PR:PROJECT_TITLE differentiation reveals subset-specific regulation of glycosphingolipid pathways PR:PROJECT_TYPE MS: Targeted analysis PR:PROJECT_SUMMARY This project is associated with five different studies(Part 1-5) and each study PR:PROJECT_SUMMARY is associated with one dataset. All the datasets are submitted to Metabolomics PR:PROJECT_SUMMARY Workbench. Part 1/5: It includes untargeted lipidomic analysis of CD4+ T-cell PR:PROJECT_SUMMARY subsets(Th1,Th2,Th17 and iTreg cells) and their paired control (Th0) cells. Part PR:PROJECT_SUMMARY 2/5: It includes quantitative targeted measurements of sphingolipids(ceramides PR:PROJECT_SUMMARY and glycosphingolipids) in Th17, iTreg, and their paired control(Th0)cells. Part PR:PROJECT_SUMMARY 3/5: It includes quantitative targeted measurements of sphingolipids(ceramides PR:PROJECT_SUMMARY and glycosphingolipids) in Th17 cells before(scrambled / control)and after the PR:PROJECT_SUMMARY triple knockdown of SPTLC1,2,3 genes (SPT de novo pathway: sphingolipid PR:PROJECT_SUMMARY metabolism). Part 4/5: It includes quantitative targeted measurements of PR:PROJECT_SUMMARY sphingolipids(ceramides, glycosphingolipids) in Th17 cells before (scrambled / PR:PROJECT_SUMMARY control)and after the knockdown of UGCG gene(GCS pathway: sphingolipid PR:PROJECT_SUMMARY metabolism). Part 5/5: It includes measurements of sphingolipids PR:PROJECT_SUMMARY (sphingomyelins)in Th17 cells before (scrambled / control) and after the PR:PROJECT_SUMMARY knockdown of UGCG gene(GCS pathway: sphingolipid metabolism). PR:INSTITUTE University of Turku PR:DEPARTMENT Systems Medicine, Turku Bioscience PR:LABORATORY Metabolomics PR:LAST_NAME Sen PR:FIRST_NAME Partho PR:ADDRESS Tykistökatu 6B, BioCity, 5th Floor, Turku, Southwest, 20521, Finland PR:EMAIL partho.sen@utu.fi PR:PHONE 0469608145 #STUDY ST:STUDY_TITLE Quantitative measurements of sphingolipids in Th17 cells before and after the ST:STUDY_TITLE knockdown of UGCG gene (GCS pathway: sphingolipid metabolism) (part-IV) ST:STUDY_TYPE MS: Targeted analysis ST:STUDY_SUMMARY Part 4/5: It includes quantitative targeted measurements of sphingolipids ST:STUDY_SUMMARY (ceramides, glycosphingolipids) in Th17 cells before (scrambled / control) and ST:STUDY_SUMMARY after the knockdown of UGCG gene (GCS pathway: sphingolipid metabolism). ST:INSTITUTE University of Turku ST:DEPARTMENT Systems Medicine, Turku Bioscience ST:LABORATORY Metabolomics ST:LAST_NAME Sen ST:FIRST_NAME Partho ST:ADDRESS Tykistökatu 6B, BioCity, 5th Floor, Turku, Southwest, 20521, Finland ST:EMAIL partho.sen@utu.fi ST:PHONE 0469608145 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Not applicable #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 - SCr-D1 Treatment:Scrambled | Types:Control Cell Type=T17; RAW_FILE_NAME=Data20201120_T Cells Batch2_diluted and undiluted-1-dil.mzML SUBJECT_SAMPLE_FACTORS - KD-D1 Treatment:UGCG-knockdown | Types:Test Cell Type=T17; RAW_FILE_NAME=Data20201120_T Cells Batch2_diluted and undiluted-2-dil.mzML SUBJECT_SAMPLE_FACTORS - SCr-D2 Treatment:Scrambled | Types:Control Cell Type=T17; RAW_FILE_NAME=Data20201120_T Cells Batch2_diluted and undiluted-3-dil.mzML SUBJECT_SAMPLE_FACTORS - KD-D2 Treatment:UGCG-knockdown | Types:Test Cell Type=T17; RAW_FILE_NAME=Data20201120_T Cells Batch2_diluted and undiluted-4-dil.mzML SUBJECT_SAMPLE_FACTORS - SCr-D3 Treatment:Scrambled | Types:Control Cell Type=T17; RAW_FILE_NAME=Data20201120_T Cells Batch2_diluted and undiluted-5-dil.mzML SUBJECT_SAMPLE_FACTORS - KD-D3 Treatment:UGCG-knockdown | Types:Test Cell Type=T17; RAW_FILE_NAME=Data20201120_T Cells Batch2_diluted and undiluted-6-dil.mzML #COLLECTION CO:COLLECTION_SUMMARY CD4+ T-cells were isolated from human umbilical cord blood as described CO:COLLECTION_SUMMARY previously (Khan et al., 2020; Tripathi et al., 2017; Ubaid et al., 2018). CD4+ CO:COLLECTION_SUMMARY T-cells were isolated from human umbilical cord blood as described previously CO:COLLECTION_SUMMARY [1-3]. 1. Ubaid, U. et al. Transcriptional Repressor HIC1 Contributes to CO:COLLECTION_SUMMARY Suppressive Function of Human Induced Regulatory T Cells. Cell Rep 22, CO:COLLECTION_SUMMARY 2094-2106, doi:10.1016/j.celrep.2018.01.070 (2018). 2. Khan, M. M. et al. CIP2A CO:COLLECTION_SUMMARY Constrains Th17 Differentiation by Modulating STAT3 Signaling. iScience 23, CO:COLLECTION_SUMMARY 100947, doi:10.1016/j.isci.2020.100947 (2020). 3. Tripathi, S. K. et al. CO:COLLECTION_SUMMARY Genome-wide Analysis of STAT3-Mediated Transcription during Early Human Th17 CO:COLLECTION_SUMMARY Cell Differentiation. Cell Rep 19, 1888-1901, doi:10.1016/j.celrep.2017.05.013 CO:COLLECTION_SUMMARY (2017). CO:SAMPLE_TYPE T-cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For Th17 cell differentiation, isolated CD4+ cells were activated with a TR:TREATMENT_SUMMARY combination of plate-bound anti-CD3 (750 ng/24-well culture plate well; TR:TREATMENT_SUMMARY Immunotech/Beckman Coulter REF # IM-1304) and soluble anti-CD28 ((1ug/mL; TR:TREATMENT_SUMMARY Immunotech/Beckman coulter REF # IM1376) antibodies in serum-free X-Vivo 20 TR:TREATMENT_SUMMARY medium (Lonza), in the absence (Th0) or presence (Th17) of IL-6 (20ng/ml, Roche, TR:TREATMENT_SUMMARY Cat# 11138600 001); IL-1β (10ng/ml, R&D Systems Cat # 201 LB); TGF-β1 TR:TREATMENT_SUMMARY (10ng/ml, R&D Systems Cat# 240); anti-IL-4 (1 g/ml) R&D Systems Cat# MAB204) TR:TREATMENT_SUMMARY and anti-IFN-γ (1 μg/ml R&D Systems Cat#MAB-285). Differentiation of Th17 TR:TREATMENT_SUMMARY cells was confirmed by measuring IL-17 expression by quantitative real-time PCR, TR:TREATMENT_SUMMARY at 72 hours of Th17 / Th0 culturing. For iTreg cell culturing, after of CD25+ TR:TREATMENT_SUMMARY cells, done using LD columns and a CD25 depletion kit (Miltenyi Biotec), TR:TREATMENT_SUMMARY CD4+CD25− cells were activated with plate-bound anti-CD3 (500 ng/24-well TR:TREATMENT_SUMMARY culture plate well) and soluble anti-CD28 (500 ng/mL) at a density of 2 × 106 TR:TREATMENT_SUMMARY cells/mL of X-vivo 15 serum-free medium (Lonza). For iTreg differentiation, the TR:TREATMENT_SUMMARY medium was supplemented with IL-2 (12 ng/mL), TGF-β (10 ng/mL) (both from R&D TR:TREATMENT_SUMMARY Systems), all-trans retinoic acid (ATRA) (10 nM; Sigma-Aldrich), and human serum TR:TREATMENT_SUMMARY (10%) and cultured at 37°C in 5% CO2. Control Th0 cells were stimulated with TR:TREATMENT_SUMMARY plate-bound anti-CD3 soluble anti-CD28 antibodies without cytokines. For TR:TREATMENT_SUMMARY confirmation of iTreg cell differentiation, we used intracellular staining to TR:TREATMENT_SUMMARY measure, at 72 hours of iTreg culturing, expression of FOXP3 which is the major TR:TREATMENT_SUMMARY transcription factor driving Treg differentiation. Intracellular staining was TR:TREATMENT_SUMMARY performed using buffer sets of Human Regulatory T-cell Staining Kit TR:TREATMENT_SUMMARY (eBioscience/Thermo Fisher Scientific), following the manufacturer’s protocol. TR:TREATMENT_SUMMARY The following antibodies were used: anti-human FOXP3-PE (eBioscience, Cat. No. TR:TREATMENT_SUMMARY 12-4776-42) and rat IgG2a isotype control (eBioscience, Cat. No. 72-4321-77A). TR:TREATMENT_SUMMARY All samples were acquired by a flow cytometer (LSRII) and analyzed either with TR:TREATMENT_SUMMARY FlowJo (FLOWJO, LLC) or with Flowing Software. For Th1 and Th2 cells, purified TR:TREATMENT_SUMMARY naive CD4+ T-cells were activated with plate-bound anti-CD3 (500 ng/24-well TR:TREATMENT_SUMMARY culture plate well) and 500 ng/ml soluble anti-CD28 and cultured in the absence TR:TREATMENT_SUMMARY (Th0) or presence of 2.5 ng/ml IL-12 (R&D Systems) (Th1) or 10 ng/ml IL-4 (R&D TR:TREATMENT_SUMMARY Systems) (for Th2). At 48 hours following the activation of the cells, 17 ng/ml TR:TREATMENT_SUMMARY IL-2 (R&D Systems) was added to the cultures. Differentiation of Th1 and Th2 TR:TREATMENT_SUMMARY cells was confirmed by measuring (using flow cytometry) the expression of T-bet TR:TREATMENT_SUMMARY and Gata3 at 72 hours after cell activation. Briefly, cells were fixed and TR:TREATMENT_SUMMARY permeabilized using the Intracellular Fixation & Permeabilization Buffer Set TR:TREATMENT_SUMMARY (eBioscience / Thermo Fisher Scientific), according the manufacturer’s TR:TREATMENT_SUMMARY protocol. The following antibodies were used: anti-human GATA3-PE (eBioscience, TR:TREATMENT_SUMMARY 12-9966), anti-human T-bet-BV711 (BD, 563320) and corresponding isotype controls TR:TREATMENT_SUMMARY (BV711 Mouse IgG1, BD, 563044 and PE Rat IgG2b, eBioscience, 12-4031-82). TR:TREATMENT_SUMMARY Samples were acquired by BD LSRFortessa™ cell analyzer and data were analyzed TR:TREATMENT_SUMMARY using FlowJo software (FLOWJO, LLC). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The frozen cell preps were defrosted on ice. The samples were extracted using a SP:SAMPLEPREP_SUMMARY modified Folch method[1]. 1. Sen, P. et al. Persistent Alterations in Plasma SP:SAMPLEPREP_SUMMARY Lipid Profiles Before Introduction of Gluten in the Diet Associated With SP:SAMPLEPREP_SUMMARY Progression to Celiac Disease. Clin Transl Gastroenterol 10, 1-10, SP:SAMPLEPREP_SUMMARY doi:10.14309/ctg.0000000000000044 (2019). Briefly, 120 µL of cold (4 °C) SP:SAMPLEPREP_SUMMARY extraction solvent (CHCl3: MeOH, (2:1 v/v) was added to the samples. The SP:SAMPLEPREP_SUMMARY extraction solvent containing the following internal standards: C17 SP:SAMPLEPREP_SUMMARY Lactosyl(beta) ceramide (D18:1/17:0, 20 ppb), C17 Glucosyl(beta) ceramide SP:SAMPLEPREP_SUMMARY (D18:1/17:0, 20 ppb), C17 ceramide (D18:1/17:0, 20 ppb), C16 ceramide-d7 SP:SAMPLEPREP_SUMMARY (d18:1-d7/16:0, 16,57 ppb), C18 ceramide-d7 (d18:1-d7/18:0, 8.75 ppb), C24 SP:SAMPLEPREP_SUMMARY ceramide-d7 (d18:1-d7/24:0, 20 ppb), and C24:1 ceramide-d7 (d18:1-d7/24:1(15Z), SP:SAMPLEPREP_SUMMARY 9,96 ppb). The samples were the vortexed briefly and left on ice for 30 minutes. SP:SAMPLEPREP_SUMMARY The samples were then centrifuged (9400g, 5 min, 4 °C) and then 60 µL of the SP:SAMPLEPREP_SUMMARY bottom layer was transfer to a clean mass spectrometry vial (2 mL). The samples SP:SAMPLEPREP_SUMMARY were then stored at –80 °C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation was performed on an ACQUITY UHPLC BEH C18 column (2.1 CH:CHROMATOGRAPHY_SUMMARY mm × 100 mm, particle size 1.7 µm, Waters, Milford, MA, USA). The flow rate CH:CHROMATOGRAPHY_SUMMARY was set at 0.4 ml/min throughout the run with an injection volume of 1 µL. The CH:CHROMATOGRAPHY_SUMMARY following solvents were used for the gradient elution: Solvent A was H2O with 1% CH:CHROMATOGRAPHY_SUMMARY NH4Ac (1M) and HCOOH (0.1%) added. Solvent B was a mixture of ACN: IPA (1:1 v/v) CH:CHROMATOGRAPHY_SUMMARY with 1% NH4Ac (1M) and HCOOH (0.1%) added. The gradient was programmed as CH:CHROMATOGRAPHY_SUMMARY follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The CH:CHROMATOGRAPHY_SUMMARY column was equilibrated with a 7min period of 35 % B prior to the next run. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME ACQUITY UHPLC CH:COLUMN_NAME Waters BEH C18 (100 × 2.1 mm, 1.7 µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The ceramides were quantified using a targeted multiple reaction monitoring MS:MS_COMMENTS (MRM) method using UHPLC as a separation technique. The LC separation was based MS:MS_COMMENTS on the global lipidomics method previously described 71. Briefly, the UHPLC was MS:MS_COMMENTS a Exion AD (Sciex) integrated system. The samples were held in a cool box at 15 MS:MS_COMMENTS °C prior to the analysis. The needle was washed with both a 10% DCM in MeOH and MS:MS_COMMENTS ACN: MeOH: IPA: H2O (1:1:1:1 v/v/v/v) with 1% HCOOH for a total of 7.5 seconds MS:MS_COMMENTS each. The solvents were delivered using a quaternary solvent and a column oven MS:MS_COMMENTS (set to 50 °C). The separation was performed on an ACQUITY UHPLC BEH C18 column MS:MS_COMMENTS (2.1 mm × 100 mm, particle size 1.7 µm, Waters, Milford, MA, USA). The flow MS:MS_COMMENTS rate was set at 0.4 ml/min throughout the run with an injection volume of 1 µL. MS:MS_COMMENTS The following solvents were used for the gradient elution: Solvent A was H2O MS:MS_COMMENTS with 1% NH4Ac (1M) and HCOOH (0.1%) added. Solvent B was a mixture of ACN: IPA MS:MS_COMMENTS (1:1 v/v) with 1% NH4Ac (1M) and HCOOH (0.1%) added. The gradient was programmed MS:MS_COMMENTS as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The MS:MS_COMMENTS column was equilibrated with a 7min period of 35 % B prior to the next run. The MS:MS_COMMENTS mass spectrometer was a Sciex 5500 QTRAP (Sciex) set in scheduled MRM mode. All MS:MS_COMMENTS lipids were identified for their fatty acid composition by MS/MS to confirm MS:MS_COMMENTS their exact identification, there was also a linear relationship between the MS:MS_COMMENTS increasing number of carbons in the lipid chain and its corresponding retention MS:MS_COMMENTS time. Due to the isobaric nature of sugars we were unable to differentiate Glc MS:MS_COMMENTS and Glc head groups. All data were integrated using the quantitation tool in MS:MS_COMMENTS MultiQuant (3.0.3), all peaks were manually checked. Any analytes which were MS:MS_COMMENTS over the concentration of the standard curve were diluted (1:25) with the same MS:MS_COMMENTS extraction solvent minus the internal standards. The quantification was MS:MS_COMMENTS performed using class-based internal standards and in the case of those ceramide MS:MS_COMMENTS species without an authentic standard in the standard curve mix, we used the MS:MS_COMMENTS closest related structure. The standard curve mixture contained: Glucosyl (beta) MS:MS_COMMENTS C12 ceramide, Lactosyl (beta)) C12 ceramide, C18 ceramide (D18:1/18:1), C18:1 MS:MS_COMMENTS dihydroceramide (d18:0/18:1(9Z)) and was run at the following levels (all in MS:MS_COMMENTS ppb): 100, 80, 60, 50, 40, 30, 20, 10 for the C12 standards and 10, 8, 6, 5, 4, MS:MS_COMMENTS 3, 2,1 for all C18 standards. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/ml MS_METABOLITE_DATA_START Samples SCr-D1 KD-D1 SCr-D2 KD-D2 SCr-D3 KD-D3 Factors Treatment:Scrambled | Types:Control Treatment:UGCG-knockdown | Types:Test Treatment:Scrambled | Types:Control Treatment:UGCG-knockdown | Types:Test Treatment:Scrambled | Types:Control Treatment:UGCG-knockdown | Types:Test Cer(d18:1/18:1(9Z)) 165.5497765 149.3901143 110.8672349 116.9300258 137.3496363 140.3680522 Cer(d18:1/12:0) 1.502376703 1.52260334 1.202910099 1.091365795 1.777951429 1.481896423 Cer(d18:1/14:0) 29.55489419 27.56669985 22.40388412 20.36479389 35.17247527 27.66640244 Cer(d18:1/16:0) 928.7356992 599.9810567 615.3534801 531.9532258 806.7984829 603.5228981 Cer(d18:1/18:0) 82.40383717 70.5810598 56.097338 40.7137742 73.55688026 42.20913603 Cer(d18:1/20:0) 52.07999865 44.52437377 35.45021951 21.87486606 39.86094705 26.49451417 Cer(d18:1/22:0) 271.5150051 248.2854708 191.7708947 139.4624282 211.4898898 202.6828705 Cer(d18:1/24:0) 602.3973041 438.4527772 373.086957 299.4081292 354.8420076 345.918128 Cer(d18:1/25:0) 7.368100413 6.806342868 6.811688153 6.085509264 6.592420736 6.347876289 Cer(d18:1/26:0) 35.5032941 35.08529927 29.42685128 19.96978429 33.46049494 27.67976419 Cer(d18:1/24:1) 245.1306557 230.5961753 187.8576079 114.2104909 224.5639202 210.1499233 Cer(d18:1/26:1) 6.973761754 7.687478744 7.054747898 7.483367857 5.19679129 7.318350182 HexCer(d18:1/12:0) 2.866755659 3.154303461 1.892208403 1.817797824 3.356721746 2.39851355 HexCer(d18:1/16:0) 1306.352186 1719.209883 1320.078348 1053.910325 1247.886116 1079.815391 HexCer(d18:1/18:0) 45.09884784 52.47159172 34.37667881 31.13068897 39.27826337 33.72610896 HexCer(d18:1/20:0) 85.12438836 109.6836897 76.57171302 67.56169209 74.39699971 52.68674862 HexCer(d18:1/22:0) 618.6237387 812.4503969 598.880169 593.1030174 648.0982202 477.5298368 HexCer(d18:1/23:0) 61.48994593 41.02773658 39.10235178 24.66569068 46.03994935 35.89060192 HexCer(d18:1/24:0) 1496.247555 1202.910019 1273.036397 770.3801448 1060.821615 847.5158402 HexCer(d18:1/24:1) 1003.649252 937.0698414 868.8034849 738.9238022 1059.996306 588.1125305 diHexCer(d18:1/12:0) 2.051358075 2.520561894 1.496249475 1.580361586 2.520422924 1.886980405 diHexCer(d18:1/14:0) 66.60393874 72.8291762 53.79193036 60.20379634 76.72191234 66.9185382 diHexCer(d18:1/16:0) 1410.557494 2423.481918 1163.653657 1365.371295 1337.2141 1531.257181 diHexCer(d18:1/18:0) 78.33555212 73.56516642 39.38482443 48.61082201 58.75182398 45.36558185 diHexCer(d18:1/20:0) 174.746267 316.5376174 131.9687709 142.1335085 149.1409446 153.0217957 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID KEGG ID Cer(d18:1/18:1(9Z)) Cer(d18:1/12:0) Cer(d18:1/14:0) Cer(d18:1/16:0) Cer(d18:1/18:0) Cer(d18:1/20:0) Cer(d18:1/22:0) Cer(d18:1/24:0) Cer(d18:1/25:0) Cer(d18:1/26:0) Cer(d18:1/24:1) Cer(d18:1/26:1) HexCer(d18:1/12:0) HexCer(d18:1/16:0) HexCer(d18:1/18:0) HexCer(d18:1/20:0) HexCer(d18:1/22:0) HexCer(d18:1/23:0) HexCer(d18:1/24:0) HexCer(d18:1/24:1) diHexCer(d18:1/12:0) diHexCer(d18:1/14:0) diHexCer(d18:1/16:0) diHexCer(d18:1/18:0) diHexCer(d18:1/20:0) METABOLITES_END #END