Summary of Study ST003135
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 PR001948. The data can be accessed directly via it's Project DOI: 10.21228/M8XF0R 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 | ST003135 |
| Study Title | Metabolomic study of T effector and T regulatory cells in severe allergic patients |
| Study Summary | Metabolism has a profound impact on T cell fate and function. Uncovering the metabolome of circulating human CD4+ T effector memory (Teff) and T regulatory (Treg) cells would enable better understanding of Th2-driven diseases, such as allergy or asthma. Here, we demonstrated that in healthy humans, energy metabolism and functions of memory CD4+ Teff cells mainly relied on amino acids, whereas Treg cells predominantly used fatty acids. Arginine and phenylalanine increased T cell receptorinduced glycolysis and oxidative phosphorylation in total and memory CD4+ T cells, but high levels of phenylalanine limited CD4+ T cell proliferation via disrupting mitochondrial respiration and activation of L-phenylalanine oxidase, IL4I1. Accordingly, lowest levels of phenylalanine were linked with the pathogenic Th2a cells, and impaired Treg cells in patients with the most severe forms of allergies. It all suggests that phenylalanine is a metabolic checkpoint of pathogenic Th2 cells development. |
| Institute | Universidad CEU San Pablo |
| Department | Química y Bioquímica |
| Last Name | Villaseñor |
| First Name | Alma |
| Address | Urbanización Montepríncipe, n/s, Boadilla del Monte, Madrid, 28003, Spain |
| alma.villasenor@ceu.es | |
| Phone | 913724750 |
| Submit Date | 2024-02-22 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-08-22 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001948 |
| Project DOI: | doi: 10.21228/M8XF0R |
| Project Title: | Metabolomic study of T effector and T regulatory cells in severe allergic patients |
| Project Type: | MS untargeted analysis combining lipidomics and metabolomics. |
| Project Summary: | Metabolism has a profound impact on T cell fate and function. Uncovering the metabolome of circulating human CD4+ T effector memory (Teff) and T regulatory (Treg) cells would enable better understanding of Th2-driven diseases, such as allergy or asthma. Here, we demonstrated that in healthy humans, energy metabolism and functions of memory CD4+ Teff cells mainly relied on amino acids, whereas Treg cells predominantly used fatty acids. Arginine and phenylalanine increased T cell receptorinduced glycolysis and oxidative phosphorylation in total and memory CD4+ T cells, but high levels of phenylalanine limited CD4+ T cell proliferation via disrupting mitochondrial respiration and activation of L-phenylalanine oxidase, IL4I1. Accordingly, lowest levels of phenylalanine were linked with the pathogenic Th2a cells, and impaired Treg cells in patients with the most severe forms of allergies. It all suggests that phenylalanine is a metabolic checkpoint of pathogenic Th2 cells development. |
| Institute: | Universidad CEU San Pablo |
| Department: | Química y Bioquímica |
| Last Name: | Villaseñor |
| First Name: | Alma |
| Address: | Urbanización Montepríncipe, n/s, Boadilla del Monte, Madrid, 28003, Spain |
| Email: | alma.villasenor@ceu.es |
| Phone: | 913724750 |
Subject:
| Subject ID: | SU003252 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | Adult patients (>18 years old) |
| Gender: | Male and female |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Experimental_Group | Cell_Type |
|---|---|---|---|---|
| SA339354 | Blank_Eff_Polar | PBMCs | Blank | Teff |
| SA339355 | Blank_Eff_Lipids | PBMCs | Blank | Teff |
| SA339356 | Blank_Reg_Polar | PBMCs | Blank | Treg |
| SA339357 | Blank_Reg_Lipids | PBMCs | Blank | Treg |
| SA339358 | Curve_1500_Eff_Lipids | PBMCs | Cell_Curve | Teff |
| SA339359 | Curve_500_Eff_Lipids | PBMCs | Cell_Curve | Teff |
| SA339360 | Curve_100_Eff_Lipids | PBMCs | Cell_Curve | Teff |
| SA339361 | Curve_250_Eff_Polar | PBMCs | Cell_Curve | Teff |
| SA339362 | Curve_250_Eff_Lipids | PBMCs | Cell_Curve | Teff |
| SA339363 | Curve_500_Eff_Polar | PBMCs | Cell_Curve | Teff |
| SA339364 | Curve_1500_Eff_Polar | PBMCs | Cell_Curve | Teff |
| SA339365 | Curve_100_Eff_Polar | PBMCs | Cell_Curve | Teff |
| SA339366 | Curve_50_Reg_Lipids | PBMCs | Cell_Curve | Treg |
| SA339367 | Curve_100_Reg_Polar | PBMCs | Cell_Curve | Treg |
| SA339368 | Curve_50_Reg_Polar | PBMCs | Cell_Curve | Treg |
| SA339369 | Curve_25_Reg_Lipids | PBMCs | Cell_Curve | Treg |
| SA339370 | Curve_10_Reg_Polar | PBMCs | Cell_Curve | Treg |
| SA339371 | Curve_10_Reg_Lipids | PBMCs | Cell_Curve | Treg |
| SA339372 | Curve_100_Reg_Lipids | PBMCs | Cell_Curve | Treg |
| SA339373 | Curve_25_Reg_Polar | PBMCs | Cell_Curve | Treg |
| SA339374 | B2_Eff_Lipids | PBMCs | Mild | Teff |
| SA339375 | B3_Eff_Polar | PBMCs | Mild | Teff |
| SA339376 | B4_Eff_Polar | PBMCs | Mild | Teff |
| SA339377 | B2_Eff_Polar | PBMCs | Mild | Teff |
| SA339378 | B3_Eff_Lipids | PBMCs | Mild | Teff |
| SA339379 | B4_Eff_Lipids | PBMCs | Mild | Teff |
| SA339380 | B4_Reg_Polar | PBMCs | Mild | Treg |
| SA339381 | B3_Reg_Lipids | PBMCs | Mild | Treg |
| SA339382 | B4_Reg_Lipids | PBMCs | Mild | Treg |
| SA339383 | B3_Reg_Polar | PBMCs | Mild | Treg |
| SA339384 | B2_Reg_Polar | PBMCs | Mild | Treg |
| SA339385 | B2_Reg_Lipids | PBMCs | Mild | Treg |
| SA339386 | A4_Eff_Polar | PBMCs | Non_Allergic | Teff |
| SA339387 | A4_Eff_Lipids | PBMCs | Non_Allergic | Teff |
| SA339388 | A7_Eff_Polar | PBMCs | Non_Allergic | Teff |
| SA339389 | A7_Eff_Lipids | PBMCs | Non_Allergic | Teff |
| SA339390 | A8_Eff_Polar | PBMCs | Non_Allergic | Teff |
| SA339391 | A5_Eff_Polar | PBMCs | Non_Allergic | Teff |
| SA339392 | A2_Eff_Lipids | PBMCs | Non_Allergic | Teff |
| SA339393 | A6_Eff_Lipids | PBMCs | Non_Allergic | Teff |
| SA339394 | A2_Eff_Polar | PBMCs | Non_Allergic | Teff |
| SA339395 | A8_Eff_Lipids | PBMCs | Non_Allergic | Teff |
| SA339396 | A6_Eff_Polar | PBMCs | Non_Allergic | Teff |
| SA339397 | A5_Eff_Lipids | PBMCs | Non_Allergic | Teff |
| SA339398 | A6_Reg_Polar | PBMCs | Non_Allergic | Treg |
| SA339399 | A7_Reg_Polar | PBMCs | Non_Allergic | Treg |
| SA339400 | A7_Reg_Lipids | PBMCs | Non_Allergic | Treg |
| SA339401 | A6_Reg_Lipids | PBMCs | Non_Allergic | Treg |
| SA339402 | A8_Reg_Polar | PBMCs | Non_Allergic | Treg |
| SA339403 | A4_Reg_Polar | PBMCs | Non_Allergic | Treg |
| SA339404 | A5_Reg_Polar | PBMCs | Non_Allergic | Treg |
| SA339405 | A8_Reg_Lipids | PBMCs | Non_Allergic | Treg |
| SA339406 | A4_Reg_Lipids | PBMCs | Non_Allergic | Treg |
| SA339407 | A5_Reg_Lipids | PBMCs | Non_Allergic | Treg |
| SA339408 | A2_Reg_Lipids | PBMCs | Non_Allergic | Treg |
| SA339409 | A2_Reg_Polar | PBMCs | Non_Allergic | Treg |
| SA339410 | QC15_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339411 | QC15_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339412 | QC16_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339413 | QC14_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339414 | QC14_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339415 | QC13_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339416 | QC12_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339417 | QC13_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339418 | QC16_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339419 | QC21_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339420 | QC20_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339421 | QC20_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339422 | QC21_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339423 | QC17_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339424 | QC19_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339425 | QC12_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339426 | QC17_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339427 | QC19_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339428 | QC18_Eff_Polar | PBMCs | Quality_Control | Teff |
| SA339429 | QC18_Eff_Lipids | PBMCs | Quality_Control | Teff |
| SA339430 | QC8_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339431 | QC7_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339432 | QC7_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339433 | QC8_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339434 | QC9_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339435 | QC6_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339436 | QC10_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339437 | QC10_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339438 | QC9_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339439 | QC3_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339440 | QC2_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339441 | QC2_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339442 | QC1_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339443 | QC1_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339444 | QC3_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339445 | QC4_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339446 | QC5_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339447 | QC5_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339448 | QC4_Reg_Lipids | PBMCs | Quality_Control | Treg |
| SA339449 | QC6_Reg_Polar | PBMCs | Quality_Control | Treg |
| SA339450 | C12_Eff_Polar | PBMCs | Severe | Teff |
| SA339451 | C6_Eff_Polar | PBMCs | Severe | Teff |
| SA339452 | C9_Eff_Lipids | PBMCs | Severe | Teff |
| SA339453 | C10_Eff_Polar | PBMCs | Severe | Teff |
Collection:
| Collection ID: | CO003245 |
| Collection Summary: | Whole blood was drawn from the participants into heparin tubes and serum in silica tubes. Next, PBMCs were isolated using Ficoll Plaque by gradient density centrifugation according to manufacturer instructions. Isolated PMBCs were then immediately frozen and kept at -80˚C until analysis. Teff and Treg cell subpopulations were determined through FlowJo X (v.10.7.1.) and FlowSOM (v.2.6). |
| Sample Type: | Blood (whole) |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003261 |
| Treatment Summary: | After cell sorting, the only treatment received was the addition of a volume of 50 uL of MeOH to each sample and then cells were frozen at -80 ºC until metabolomics and lipidomic analysis. |
Sample Preparation:
| Sampleprep ID: | SP003259 |
| Sampleprep Summary: | Frozen cells in MeOH were thawed at 4˚C. Then a first extraction targeted at lipid metabolites was performed by adding MTBE to a final concentration of MTBE: MeOH (1:4). Samples were then sonicated for a total of 15 min at 15W (3 rounds of 5 min sonication), thoroughly vortexed, and centrifuged for 10 min at 16000 g to remove any debris. The supernatant was collected. Then a second extraction took place to obtain the most polar metabolites in the sample. To that extent, 100 μl of a mixture of H2O:MeOH (1:4) was added and the sample was sonicated again for a total of 15 min at 15W (3 rounds of 5 min sonication), thoroughly vortexed and centrifuged for 10 min at 16000 g to remove any debris. The supernatant was collected. Polar samples were kept at 4 degrees until their analysis. Extraction solvents were used as a blank and followed the same procedure as the samples. Quality control (QC) samples were prepared by pooling equal volumes of samples from the same cell type separately. Additionally, to select features produced only by cells, a calibration curve was made of Teff or Treg cells, respectively. Samples were divided into 2 groups depending on the type of subpopulation they belong to either Teff or Treg, and randomized. |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN005145 | AN005146 | AN005147 | AN005148 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase | Reversed phase |
| Chromatography system | Agilent 1290 Infinity II | Agilent 1290 Infinity II | Agilent 1290 Infinity II | Agilent 1290 Infinity II |
| Column | Agilent ZORBAX RRHD Extend-C18 (50 x 2.1mm,1.8um) | Agilent ZORBAX RRHD Extend-C18 (50 x 2.1mm,1.8um) | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | QTOF | QTOF | QTOF | QTOF |
| MS instrument name | Agilent 6550 QTOF | Agilent 6550 QTOF | Agilent 6550 QTOF | Agilent 6550 QTOF |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
| Units | Peak area | Peak area | Peak area | Peak area |
Chromatography:
| Chromatography ID: | CH003895 |
| Chromatography Summary: | For polar metabolites separation, 2 μL of the sample were injected into a Zorbax Extend C18 (4.6 × 50 mm, 1.8 μm; Agilent, Waldbronn, Germany), with a guard column Zorbax Extend C18 (3 × 5 mm, 1.8 μm; Agilent), both maintained at 60 °C. The flow rate was set at 0.6 mL/min. The elution gradient involved a mobile phase consisting of: (A) water containing 0.1% of formic acid and (B) acetonitrile containing 0.1% of formic acid. The initial conditions were set at 5% phase B for 1 min, which increased linearly to 80% phase B in 7 min. Then in 4.5 min it increased until 100% of phase. Then the equipment returned to the initial conditions in 0.5 min, which were held for 3 min for column reconditioning. |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Agilent ZORBAX RRHD Extend-C18 (50 x 2.1mm,1.8um) |
| Column Temperature: | 60 ºC |
| Flow Gradient: | 0-1 min: 5% B; 1-8 min: linear increase until 80% B; 8-12.5 min: linear increase until 100% B. Then the equipment returned to the initial conditions in 0.5 min, which were held for 3 min for column reconditioning. |
| Flow Rate: | 0.6 mL/min |
| Solvent A: | 100% water; 0.1% of formic acid |
| Solvent B: | 100% acetonitrile; 0.1% of formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003896 |
| Chromatography Summary: | For the lipidic extraction, the Agilent 1290 Infinity II Multisampler system, equipped with a multi-wash option, was used to uptake 1 and 2 µL of extracted samples in positive and negative ionization modes, respectively. The multisampler temperature was maintained at 15 °C to preserve lipids in a stable environment and avoid precipitation. An Agilent InfinityLab Poroshell 120 ECsingle bondC18 (3.0 × 100 mm, 2.7 µm) (Agilent Technologies) column and a compatible guard column (Agilent InfinityLab Poroshell 120 ECsingle bondC18, 3.0 × 5 mm, 2.7 µm) were used and maintained at 50 °C. The chromatography gradient started at 70% of B at 0 – 1 min, 86% at 3.5 – 10 min, 100% B at 11–17 min. The starting conditions were recovered by minute 17, followed by a 2 min re-equilibration time; the total running time was 19 min. The mobile phases used for both positive and negative ionization modes consisted of (A) 10 mM ammonium acetate, 0.2 mM ammonium fluoride in 9:1 water/methanol and (B) 10 mM ammonium acetate, 0.2 mM ammonium fluoride in 2:3:5 acetonitrile/methanol/isopropanol. The flow rate was held constant, set at 0.6 mL/min. The multi-wash strategy consisted of a mixture of methanol:isopropanol (50:50, v/v) with the wash time set at 15 s, and aqueous phase:organic phase (30:70, v/v) mixture to assist in the starting conditions. |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) |
| Column Temperature: | 50 ºC |
| Flow Gradient: | 0-1 min: 70% B; 1-3.5 min: linear increase until 86% B; 3.5-10 min: 86% B; 10-11 min: linear increase until 100% B; 11-17 min: 100% B. Then the equipment returned to the initial conditions in 0.1 min, which were held for 1.9 min for column reconditioning. |
| Flow Rate: | 0.6 mL/min |
| Solvent A: | water/methanol (9/1); 10 mM ammonium acetate; 0.2 mM ammonium fluoride |
| Solvent B: | acetonitrile/methanol/isopropanol (2/3/5); 10 mM ammonium acetate; 0.2 mM ammonium fluoride |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004881 |
| Analysis ID: | AN005145 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The Agilent 6550 QTOF mass spectrometer equipped with a dual AJS ESI ion source was set with the following parameters: The capillary voltage was set at 3000 for both polarities. The drying gas flow rate was 12 L/min at 250 °C and gas nebulizer at 52 psi; fragmentor voltage was set at 175 V in ESI+ and 250 in ESI-; skimmer and octupole radio frequency voltages were set to 65 and 750 V, respectively. MS spectra were collected in the centroid mode at a scan rate of 3 spectra/s. The MS detection window was performed in a full scan from 100 to 1200 m/z for both modes. Automatic MS recalibration during batch analysis was carried out by introducing a reference standard into the source via a reference sprayer valve. Reference masses for ESI+ were purine (m/z = 121.0508) and HP-0921 (m/z = 922.0097). |
| Ion Mode: | POSITIVE |
| MS ID: | MS004882 |
| Analysis ID: | AN005146 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The Agilent 6550 QTOF mass spectrometer equipped with a dual AJS ESI ion source was set with the following parameters: The capillary voltage was set at 3000 for both polarities. The drying gas flow rate was 12 L/min at 250 °C and gas nebulizer at 52 psi; fragmentor voltage was set at 175 V in ESI+ and 250 in ESI-; skimmer and octupole radio frequency voltages were set to 65 and 750 V, respectively. MS spectra were collected in the centroid mode at a scan rate of 3 spectra/s. The MS detection window was performed in a full scan from 100 to 1200 m/z for both modes. Automatic MS recalibration during batch analysis was carried out by introducing a reference standard into the source via a reference sprayer valve. Reference masses for ESI - were TFA NH4 (m/z = 112.9855) and HP-0921 (m/z = 966.0007). |
| Ion Mode: | NEGATIVE |
| MS ID: | MS004883 |
| Analysis ID: | AN005147 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The Agilent 6550 QTOF mass spectrometer equipped with a dual AJS ESI ion source was set with the following parameters: 150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in centroid in mode positive and negative ESI modes in separate runs, operated in full scan mode from 50 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was used throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098 for the positive. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004884 |
| Analysis ID: | AN005148 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The Agilent 6550 QTOF mass spectrometer equipped with a dual AJS ESI ion source was set with the following parameters: 150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in centroid in mode positive and negative ESI modes in separate runs, operated in full scan mode from 50 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was used throughout the whole analysis: purine (C5H4N4) at m/z 119.0363 and HP-0921 (C18H18O6N3P3F24) at m/z 980.0163 (HP-0921+acetate) for the negative ionization modes. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction. |
| Ion Mode: | NEGATIVE |
