Summary of Study ST001676
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001078. The data can be accessed directly via it's Project DOI: 10.21228/M8C111 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001676 |
| Study Title | Lipidomic analysis of CD4+ T-cell subsets (Th1,Th2,Th17 and iTreg cells) (part I) |
| Study Type | MS, untargeted cell-based lipidomics |
| Study Summary | Part 1/5: It includes lipidomic analysis of CD4+ T-cell subsets(Th1,Th2,Th17 and iTreg cells)and their paired controls(Th0 cells). |
| Institute | University of Turku |
| Department | Systems Medicine, Turku Bioscience |
| Laboratory | Systems Medicine |
| Last Name | Sen |
| First Name | Partho |
| Address | Tykistökatu 6B, BioCity, 5th Floor, Turku, Southwest, 20521, Finland |
| partho.sen@utu.fi | |
| Phone | 0469608145 |
| Submit Date | 2021-01-28 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-11-02 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001078 |
| Project DOI: | doi: 10.21228/M8C111 |
| Project Title: | Quantitative analysis and genome-scale modeling of human CD4+ T-cell differentiation reveals subset-specific regulation of glycosphingolipid pathways |
| Project Type: | MS: Targeted analysis |
| Project Summary: | This project is associated with five different studies(Part 1-5) and each study is associated with one dataset. All the datasets are submitted to Metabolomics Workbench. Part 1/5: It includes untargeted lipidomic analysis of CD4+ T-cell subsets (Th1,Th2,Th17 and iTreg cells) and their paired control (Th0) cells. Part 2/5: It includes quantitative targeted measurements of sphingolipids (ceramides and glycosphingolipids) in Th17, iTreg, and their paired control (Th0) cells. Part 3/5: It includes quantitative targeted measurements of sphingolipids (ceramides and glycosphingolipids) in Th17 cells before (scrambled / control) and after the triple knockdown of SPTLC1,2,3 genes (SPT de novo pathway: sphingolipid metabolism). Part 4/5: It includes quantitative targeted measurements of sphingolipids (ceramides, glycosphingolipids) in Th17 cells before (scrambled / control) and after the knockdown of UGCG gene (GCS pathway: sphingolipid metabolism). Part 5/5: It includes measurements of sphingolipids (sphingomyelins) in Th17 cells before (scrambled / control) and after the knockdown of UGCG gene(GCS pathway: sphingolipid metabolism). |
| Institute: | University of Turku |
| Department: | Systems Medicine |
| Laboratory: | Metabolomics |
| Last Name: | Sen |
| First Name: | Partho |
| Address: | Tykistökatu 6B, BioCity, 5th Floor, Turku, Southwest, 20521, Finland |
| Email: | partho.sen@utu.fi |
| Phone: | 0469608145 |
Subject:
| Subject ID: | SU001753 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Not applicable |
| Cell Counts: | 5M |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Donors | Cell Type | Treatment |
|---|---|---|---|---|
| SA154504 | Th0_(Th17)_D1 | D1 | Th0_(Th17) | Activated |
| SA154503 | Th0_(Th1/Th2)_D1 | D1 | Th0_(Th1/Th2) | Activated |
| SA154505 | Th0_(Treg)_D1 | D1 | Th0_(Treg) | Activated |
| SA154506 | Th17_D1 | D1 | Th17 | Differentiated |
| SA154507 | Th1_D1 | D1 | Th1 | Differentiated |
| SA154508 | Th2_D1 | D1 | Th2 | Differentiated |
| SA154509 | Thp_CD25_D1 | D1 | Thp_CD25 | T-naive |
| SA154510 | Thp_D1 | D1 | Thp | T-naive |
| SA154511 | Treg_D1 | D1 | Treg | Differentiated |
| SA154513 | Th0_(Th17)_D2 | D2 | Th0_(Th17) | Activated |
| SA154512 | Th0_(Th1/Th2)_D2 | D2 | Th0_(Th1/Th2) | Activated |
| SA154514 | Th0_(Treg)_D2 | D2 | Th0_(Treg) | Activated |
| SA154515 | Th17_D2 | D2 | Th17 | Differentiated |
| SA154516 | Th1_D2 | D2 | Th1 | Differentiated |
| SA154517 | Th2_D2 | D2 | Th2 | Differentiated |
| SA154518 | Thp_CD25_D2 | D2 | Thp_CD25 | T-naive |
| SA154519 | Thp_D2 | D2 | Thp | T-naive |
| SA154520 | Treg_D2 | D2 | Treg | Differentiated |
| SA154522 | Th0_(Th17)_D3 | D3 | Th0_(Th17) | Activated |
| SA154521 | Th0_(Th1/Th2)_D3 | D3 | Th0_(Th1/Th2) | Activated |
| SA154523 | Th0_(Treg)_D3 | D3 | Th0_(Treg) | Activated |
| SA154524 | Th17_D3 | D3 | Th17 | Differentiated |
| SA154525 | Th1_D3 | D3 | Th1 | Differentiated |
| SA154526 | Th2_D3 | D3 | Th2 | Differentiated |
| SA154527 | Thp_D3 | D3 | Thp | T-naive |
| SA154528 | Treg_D3 | D3 | Treg | Differentiated |
| SA154530 | Th0_(Th17)_D4 | D4 | Th0_(Th17) | Activated |
| SA154529 | Th0_(Th1/Th2)_D4 | D4 | Th0_(Th1/Th2) | Activated |
| SA154531 | Th0_(Treg)_D4 | D4 | Th0_(Treg) | Activated |
| SA154532 | Th17_D4 | D4 | Th17 | Differentiated |
| SA154533 | Th1_D4 | D4 | Th1 | Differentiated |
| SA154534 | Th2_D4 | D4 | Th2 | Differentiated |
| SA154535 | Thp_CD25_D4 | D4 | Thp_CD25 | T-naive |
| SA154536 | Thp_D4 | D4 | Thp | T-naive |
| SA154537 | Treg_D4 | D4 | Treg | Differentiated |
| SA154539 | Th0_(Th17)_D5 | D5 | Th0_(Th17) | Activated |
| SA154538 | Th0_(Th1/Th2)_D5 | D5 | Th0_(Th1/Th2) | Activated |
| SA154540 | Th0_(Treg)_D5 | D5 | Th0_(Treg) | Activated |
| SA154541 | Th17_D5 | D5 | Th17 | Differentiated |
| SA154542 | Th1_D5 | D5 | Th1 | Differentiated |
| SA154543 | Th2_D5 | D5 | Th2 | Differentiated |
| SA154544 | Thp_CD25_D5 | D5 | Thp_CD25 | T-naive |
| SA154545 | Thp_D5 | D5 | Thp | T-naive |
| SA154546 | Treg_D5 | D5 | Treg | Differentiated |
| SA154547 | m/z | mass / charge | - | - |
| SA154548 | RT | retention time | - | - |
| Showing results 1 to 46 of 46 |
Collection:
| Collection ID: | CO001746 |
| Collection Summary: | CD4+ T-cells were isolated from human umbilical cord blood as described previously[1-3]. 1. Ubaid, U. et al. Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells. Cell Rep 22, 2094-2106, doi:10.1016/j.celrep.2018.01.070 (2018). 2. Khan, M. M. et al. CIP2A Constrains Th17 Differentiation by Modulating STAT3 Signaling. iScience 23, 100947, doi:10.1016/j.isci.2020.100947 (2020). 3. Tripathi, S. K. et al. Genome-wide Analysis of STAT3-Mediated Transcription during Early Human Th17 Cell Differentiation. Cell Rep 19, 1888-1901, doi:10.1016/j.celrep.2017.05.013 (2017). |
| Collection Protocol Filename: | parthosen_20210128_003828_PR_CO_Cell_Prep_Protocol.pdf |
| Sample Type: | T-cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001766 |
| Treatment Summary: | For Th17 cell differentiation, isolated CD4+ cells were activated with a combination of plate-bound anti-CD3 (750 ng/24-well culture plate well; Immunotech/Beckman Coulter REF # IM-1304) and soluble anti-CD28 ((1ug/mL; Immunotech/Beckman coulter REF # IM1376) antibodies in serum-free X-Vivo 20 medium (Lonza), in the absence (Th0) or presence (Th17) of IL-6 (20ng/ml, Roche, Cat# 11138600 001); IL-1β (10ng/ml, R&D Systems Cat # 201 LB); TGF-β1 (10ng/ml, R&D Systems Cat# 240); anti-IL-4 (1 g/ml) R&D Systems Cat# MAB204) and anti-IFN-γ (1 μg/ml R&D Systems Cat#MAB-285). Differentiation of Th17 cells was confirmed by measuring IL-17 expression by quantitative real-time PCR, at 72 hours of Th17 / Th0 culturing. For iTreg cell culturing, after of CD25+ cells, done using LD columns and a CD25 depletion kit (Miltenyi Biotec), CD4+CD25− cells were activated with plate-bound anti-CD3 (500 ng/24-well culture plate well) and soluble anti-CD28 (500 ng/mL) at a density of 2 × 106 cells/mL of X-vivo 15 serum-free medium (Lonza). For iTreg differentiation, the medium was supplemented with IL-2 (12 ng/mL), TGF-β (10 ng/mL) (both from R&D Systems), all-trans retinoic acid (ATRA) (10 nM; Sigma-Aldrich), and human serum (10%) and cultured at 37°C in 5% CO2. Control Th0 cells were stimulated with plate-bound anti-CD3 soluble anti-CD28 antibodies without cytokines. For confirmation of iTreg cell differentiation, we used intracellular staining to measure, at 72 hours of iTreg culturing, expression of FOXP3 which is the major transcription factor driving Treg differentiation. Intracellular staining was performed using buffer sets of Human Regulatory T-cell Staining Kit (eBioscience/Thermo Fisher Scientific), following the manufacturer’s protocol. The following antibodies were used: anti-human FOXP3-PE (eBioscience, Cat. No. 12-4776-42) and rat IgG2a isotype control (eBioscience, Cat. No. 72-4321-77A). All samples were acquired by a flow cytometer (LSRII) and analyzed either with FlowJo (FLOWJO, LLC) or with Flowing Software. For Th1 and Th2 cells, purified naive CD4+ T-cells were activated with plate-bound anti-CD3 (500 ng/24-well culture plate well) and 500 ng/ml soluble anti-CD28 and cultured in the absence (Th0) or presence of 2.5 ng/ml IL-12 (R&D Systems) (Th1) or 10 ng/ml IL-4 (R&D Systems) (for Th2). At 48 hours following the activation of the cells, 17 ng/ml IL-2 (R&D Systems) was added to the cultures. Differentiation of Th1 and Th2 cells was confirmed by measuring (using flow cytometry) the expression of T-bet and Gata3 at 72 hours after cell activation. Briefly, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience / Thermo Fisher Scientific), according the manufacturer’s protocol. The following antibodies were used: anti-human GATA3-PE (eBioscience, 12-9966), anti-human T-bet-BV711 (BD, 563320) and corresponding isotype controls (BV711 Mouse IgG1, BD, 563044 and PE Rat IgG2b, eBioscience, 12-4031-82). Samples were acquired by BD LSRFortessa™ cell analyzer and data were analyzed using FlowJo software (FLOWJO, LLC). |
| Treatment Protocol Filename: | parthosen_20210128_003828_PR_TR_Treatment_Summary_siRNA_KD.pdf |
Sample Preparation:
| Sampleprep ID: | SP001759 |
| Sampleprep Summary: | The samples were randomized and extracted using a modified version of the previously-published Folch procedure. Briefly, 150 µL of 0.9% NaCl was added to cell pellets, and samples then vortexed and ultrasonicated for 3 minutes. Next, 20 µL of the cell suspension was mixed with 150 µL of the 2.5 µg mL-1 internal standards solution in ice-cold CHCl3:MeOH (2:1, v/v). The internal standard solution contained the following compounds: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE (17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), 1,2-diheptadeca-noyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)). These were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). In addition, triheptadecanoin (TG(17:0/17:0/17:0)) was purchased from (Larodan AB, (Solna, Sweden). The samples were vortexed and incubated on ice for 30 min after which they were centrifuged at 7800 × g for 5 min. Finally, 60 µL from the lower layer of each sample was collected and mixed with 60 µL of ice cold CHCl3:MeOH (2:1, v/v) in LC vial. The total protein content in cells was measured by the Bradford method. |
| Sampleprep Protocol Filename: | parthosen_20210128_003828_PR_CO_Cell_Prep_Protocol.pdf |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN002734 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity UHPLC UNSPSC 41115709 |
| Column | Waters BEH C18 (00mm x 2.1mm,1.7um ) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6540 QTOF |
| Ion Mode | POSITIVE |
| Units | Intensities |
Chromatography:
| Chromatography ID: | CH002020 |
| Chromatography Summary: | Chromatographic separation was performed on an ACQUITY UHPLC BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm, Waters, Milford, MA, USA). The flow rate was set at 0.4 ml/min throughout the run with an injection volume of 1 µL. The following solvents were used for the gradient elution: Solvent A was H2O with 1% NH4Ac (1M) and HCOOH (0.1%) added. Solvent B was a mixture of ACN: IPA (1:1 v/v) with 1% NH4Ac (1M) and HCOOH (0.1%) added. The gradient was programmed as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The column was equilibrated with a 7min period of 35 % B prior to the next run. |
| Methods Filename: | parthosen_20210128_003828_PR_CH_Chromatography_metadata.pdf |
| Instrument Name: | Waters Acquity UHPLC UNSPSC 41115709 |
| Column Name: | Waters BEH C18 (00mm x 2.1mm,1.7um ) |
| Flow Gradient: | The gradient was programmed as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The column was equilibrated with a 7min period of 35 % B prior to the next run. |
| Flow Rate: | 0.4ml/min |
| Solvent A: | 100% water; 0.1% formic acid; 1% ammonium acetate |
| Solvent B: | 50% acetonitrile/50% isopropanol; 0.1% formic acid; 1% ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002531 |
| Analysis ID: | AN002734 |
| Instrument Name: | Agilent 6540 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The UHPLC-QTOFMS analyses were done with some modifications on two separate instruments. The initial lipidomic results were acquired on a UHPLC-QTOFMS system from Agilent Technologies (Santa Clara, CA, USA) combining a 1290 Infinity LC system and 6545 quadrupole time of flight mass spectrometer (QTOFMS), interfaced with a dual jet stream electrospray (dual ESI) ion source. MassHunter B.06.01 software (Agilent Technologies, Santa Clara, CA, USA) was used for all data acquisition. The SM results for UGCG-silenced Th17 cells data was acquired on a UHPLC-QTOF system from Bruker (Bruker, Billerica, MA, USA) combining an Elute UHPLC binary pump and an Impact II system QTOF system. The samples for this experiments were the same extracts that the ceramide (Cer) data was acquired from and had SM(18:1/17:0) spiked in prior to acquisition. The data was acquired using the Hystar suite of software. MZmine 2 was used for all the untargeted data processing. MS data were processed using the open source software MZmine 2.53. The following data processing steps were applied to the raw MS data: (1) Crop filtering with a m/z range of 350 – 1200 m/z and a retention time (RT) range of 2 to 15 minutes; (2) Mass detection with a noise level of 900; (3) Chromatogram builder with a min time span of 0.08 minutes, minimum height of 900 and m/z tolerance of 0.006 m/z or 10.0 ppm; (4) Chromatogram deconvolution using the local minimum search algorithm with a 70% chromatographic threshold, 0.05 min minimum RT range, 5% minimum relative height, 1200 minimum absolute height, a minimum ration of peak top/edge of 1.2 and a peak duration range of 0.08 - 1.01 minutes; (5) Isotopic peak grouper with a m/z tolerance of 5.0 ppm, RT tolerance of 0.05 minute, maximum charge of 2 and with the most intense isotope set as the representative isotope; (6) Join aligner with m/z tolerance of 0.009 or 10.0 ppm and a weight of 2, RT tolerance of 0.1 minute and a weight of 1 and with no requirement of charge state or ID and no comparison of isotope pattern; (7) Peak list row filter with a minimum of 7 peaks in a row (10% of the samples); (8) Gap filling using the same RT and m/z range gap filler algorithm with an m/z tolerance of 0.009 m/z or 11.0 ppm; (9) Identification of lipids using a custom database (based on UHPLC-MS/MS data using the same lipidomics protocol, with RT data and MS and MS/MS) search with an m/z tolerance of 0.009 m/z or 10.0 ppm and a RT tolerance of 0.2 min. In general, lipids were identified at the total number of carbons and double bonds in the structure as there was insufficient evidence to assign the specific acyl chains. Where the acyl chains are identified these have been confirmed with MS/MS level experiments and/or authentic standards. (10) Normalization using internal standards (PE (17:0/17:0), SM (d18:1/17:0), Cer (d18:1/17:0), LPC (17:0), TG (17:0/17:0/17:0) and PC (16:0/d30/18:1)) for identified lipids and closest internal standard (based on RT) for the unknown lipids, followed by calculation of the concentrations based on lipid-class calibration curves. Identification of lipids was done using an in-house spectral library with MS (and retention time), MS/MS information, and by searching the LIPID MAPS spectral database (http://www.lipidmaps.org). MS/MS data were acquired in both negative and positive ion modes in order to maximize identification coverage. Additionally, some lipids were verified by injection of commercial standards. The identification was carried out in pooled cell extracts. The peak area obtained for each lipid was normalized with lipid-class specific internal standards and with total content of protein. A (semi) quantitation was performed using lipid-class specific calibration curves. Pooled cell extracts were used for quality control, in addition to in-house plasma. The raw variation of the peak areas of internal standards in the samples was on average 15.3% and the RSD of retention times of identified lipids across all samples was on average 0.28%. The RSD of the concentrations of the identified lipids in QC samples and pooled extracts was on average 17.7%. |
| Ion Mode: | POSITIVE |
