Summary of Study ST004093
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 PR002571. The data can be accessed directly via it's Project DOI: 10.21228/M89V6K This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST004093 |
| Study Title | Autoimmune Disease Risk Gene ANKRD55 Promotes T Cell Proliferation and Th17 2 Effector Function Through Metabolic Modulation |
| Study Type | TH17 Cell cultures |
| Study Summary | ANKRD55, a gene linked to various autoimmune diseases through Genome-wide association studies (GWAS), plays a critical role in T cell function and inflammation. This study shows that Ankrd55-deficient mice are protected from T cell-mediated colitis but more vulnerable to Citrobacter rodentium infection, due to impaired CD4⁺ T cell proliferation and reduced TH17 cytokine production. To define the alterations in cellular metabolism caused by a Ankrd55-/- mutation in TH17 cells, we applied four metabolic profiling methods to in vitro differentiated TH17 cells. We found nucleotides were elevated in Ankrd55-/- compared to WT TH17 cells, with one-carbon units’ provider-folate (10-formyl-THF) being enriched. This was in accordance with the upregulated expression of enzymes in the SGOC pathway observed in Ankrd55-/- cells. |
| Institute | Broad Institute of MIT and Harvard |
| Last Name | Xavier |
| First Name | Ramnik |
| Address | 415 Main Street |
| rxavier@broadinstitute.org | |
| Phone | +1 (617) 714-7379 |
| Submit Date | 2025-07-28 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-08-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002571 |
| Project DOI: | doi: 10.21228/M89V6K |
| Project Title: | Metabolite profiling of Ankrd55-/- TH17 Cells |
| Project Summary: | Genome-wide association studies (GWAS) have linked the locus encoding Ankyrin Repeat Domain 55 (ANKRD55) with numerous autoimmune diseases; however, its biological function and role in inflammation are unclear. Here, we demonstrate that Ankrd55-deficient mice are protected from T cell-mediated colitis but are more susceptible to Citrobacter rodentium infection. Mechanistically, Ankrd55 deletion impairs CD4+ T cell proliferation and reduces effector cytokine production in T helper 17 (TH17) cells in a cell-intrinsic manner. ANKRD55 is associated with mitochondria, and its loss is associated with impaired mitochondrial respiration and activation of the LKB1 pathway. Consistently, IL-17 production can be rescued by the deletion of LKB1 in Ankrd55-deficient T cells. Altogether, our study implicates the protein ANKRD55 as a functional modulator of T cell metabolism that directly impacts TH17 responses, highlighting it as a potential target across multiple autoimmune diseases. |
| Institute: | Broad Institute of MIT and Harvard |
| Last Name: | Xavier |
| First Name: | Ramnik |
| Address: | 415 Main Street |
| Email: | rxavier@broadinstitute.org |
| Phone: | +1 (617) 714-7379 |
| Project Comments: | Jinjin Xu, Lingjia Kong, Elizabeth A. Creasey, Sneha Rath, Lei Deng, Julian Avila-Pacheco, Chenhao Li, Blayne A. Oliver, Tyler Dao, Angela R. Shih, Mark J. Daly, Alex K. Shalek, Clary B. Clish, Daniel B. Graham, Jacques Deguine |
Subject:
| Subject ID: | SU004239 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Cell Strain Details: | Naïve CD4+ T cells from either wild-type or Ankrd55-deficient C57BL/6 animals |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Genotype | Treatment |
|---|---|---|---|---|
| SA474137 | 12 | T-cells | ankrd55 ko | 25ng/mL IL-6 and 2ng/mL TGF-b1 |
| SA474138 | 10 | T-cells | ankrd55 ko | 25ng/mL IL-6 and 2ng/mL TGF-b1 |
| SA474139 | 11 | T-cells | ankrd55 ko | 25ng/mL IL-6 and 2ng/mL TGF-b1 |
| SA474140 | 5 | T-cells | ankrd55 ko | No added cytokines |
| SA474141 | 6 | T-cells | ankrd55 ko | No added cytokines |
| SA474142 | 4 | T-cells | ankrd55 ko | No added cytokines |
| SA474127 | PREFA02 | T-cells | NA | QC-drift_correction |
| SA474128 | PREFA01 | T-cells | NA | QC-drift_correction |
| SA474129 | PREFB02 | T-cells | NA | QC-pooled_ref |
| SA474130 | PREFB01 | T-cells | NA | QC-pooled_ref |
| SA474131 | 9 | T-cells | WT | 25ng/mL IL-6 and 2ng/mL TGF-b1 |
| SA474132 | 8 | T-cells | WT | 25ng/mL IL-6 and 2ng/mL TGF-b1 |
| SA474133 | 7 | T-cells | WT | 25ng/mL IL-6 and 2ng/mL TGF-b1 |
| SA474134 | 2 | T-cells | WT | No added cytokines |
| SA474135 | 3 | T-cells | WT | No added cytokines |
| SA474136 | 1 | T-cells | WT | No added cytokines |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO004232 |
| Collection Summary: | Three biological replicates were generated for each WT and Ankrd55-/- cell lines. Naïve CD4+ T cells from either wild-type or Ankrd55-/- C57BL/6 animals were isolated by using EasySep Mouse Naïve CD4+ T Cell isolation Kit (STEMCELL technologies, Vancouver Canada) and 1 million/mL of cells was cultured in 2 μg/mL anti-CD3/anti-CD28-coated 96-well plates under different cytokine conditions for 3 days. Cells were harvested on day 3 and pellets were snap-frozen and preserved at -80C until extraction. A polar extract was prepared using 1,800,000 cells extracted in 80% methanol, while a lipid extract was obtained from 600,000 cells using 100% isopropanol. The cleared extracts were analyzed with a combination of four previously described liquid chromatography high-resolution mass spectrometry (LC-MS) methods. The polar methanol extract was analyzed using the HILIC-pos (10 μL), HILIC-neg (30 μL), and C18-neg (30 μL) methods, which target polar metabolites and those of intermediate polarity. In contrast, the isopropanol extract was profiled using the C8-pos lipid profiling method. |
| Sample Type: | T-cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR004248 |
| Treatment Summary: | Cells were treated with different cytokine conditions for three days: Th0: no added cytokines. Th17n: 25ng/mL IL-6 and 2ng/mL TGF-b1. |
Sample Preparation:
| Sampleprep ID: | SP004245 |
| Sampleprep Summary: | LC–MS samples were profiled in each LC-MS method as follows: - HILIC-pos: Extracts (10 μL) were extracted with the addition of nine volumes of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories). The samples were centrifuged (10 min, 9,000g, 4°C), and the supernatants (10 μL) injected directly onto column. - C8-pos: Extracts (10 μL) using 190 μL isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL). After centrifugation (10 min, 9,000g, ambient temperature), supernatants (2 μL) were injected directly onto column. - C18-neg: Extracts (30 μL) were extracted using 90 μl methanol containing 15R-15-methyl Prostaglandin A2,15R-15-methyl Prostaglandin F2α, 15S-15-methyl Prostaglandin D2, 15S-15-methyl Prostaglandin E1, and 15S-15-methyl Prostaglandin E2 as internal standards (Cayman Chemical Co.) and centrifuged (10 min, 9,000g, 4°C). The supernatants (10 μL) were injected onto column. - HILIC-neg: Extracts (30 μL) were extracted with the addition of four volumes of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories). The samples were centrifuged (10 min, 9,000g, 4°C) and the supernatants 10 μL) were injected directly onto column. |
Combined analysis:
| Analysis ID | AN006783 | AN006784 | AN006785 | AN006786 |
|---|---|---|---|---|
| Chromatography ID | CH005151 | CH005152 | CH005153 | CH005154 |
| MS ID | MS006482 | MS006483 | MS006484 | MS006485 |
| Analysis type | MS | MS | MS | MS |
| Chromatography type | HILIC | Reversed phase | HILIC | Reversed phase |
| Chromatography system | Shimadzu Nexera X2 | Shimadzu Nexera X2 | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
| Column | Waters Atlantis HILIC (150 x 2 mm, 3 µm) | Waters Acquity BEH C8 (100 x 2.1 mm, 1.7 µm) | Phenomenex Luna NH2 (150 x 2 mm, 5 um) | Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | IDX | Orbitrap | IDX |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | POSITIVE | NEGATIVE | NEGATIVE |
| Units | Abudances | Abudances | Abudances | Abudances |
Chromatography:
| Chromatography ID: | CH005151 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters Atlantis HILIC (150 x 2 mm, 3 µm) |
| Column Temperature: | 30°C |
| Flow Gradient: | Chromatographic separation was performed using an isocratic elution at a flow rate of 250 μL/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 1 minute. This was followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes. At 10 minutes, the gradient was returned to initial isocratic conditions (5% mobile phase A) and held until 18 minutes, at which point MS acquisition was stopped. The column was equilibrated with 5% mobile phase A at a flow rate of 400 μL/min for 12 minutes, followed by a reduction to the initial flow rate of 250 μL/min for 2 minutes before the next injection. |
| Flow Rate: | 250 µL/min - 400 μL/min |
| Internal Standard: | L-Phenylalanine-d8 (CIL, DLM-372-1), L-Valine-d8 (Sigma, 486027) |
| Solvent A: | 100% Water; 10 mM Ammonium formate; 0.1% Formic acid |
| Solvent B: | 100% Acetonitrile; 0.1% Formic acid |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH005152 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters Acquity BEH C8 (100 x 2.1 mm, 1.7 µm) |
| Column Temperature: | 40°C |
| Flow Gradient: | The column was initially eluted isocratically at 450 μL/min with 80% mobile phase A for 1 minute. A linear gradient was then applied to reach 80% mobile phase B over 2 minutes, followed by a further gradient to 100% mobile phase B over 7 minutes. The system was held at 100% mobile phase B for 3 minutes before re-equilibrating at 80% mobile phase A for 9 minutes. Data acquisition ended at 15 minutes. |
| Flow Rate: | 450 µL/min |
| Internal Standard: | 12:0-12:0 PC (Avanti 850335C) |
| Solvent A: | 95% Water/5% Methanol; 10 mM Ammonium acetate; 0.1% Acetic acid |
| Solvent B: | 100% Methanol; 0.1% Acetic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH005153 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Phenomenex Luna NH2 (150 x 2 mm, 5 um) |
| Column Temperature: | 30°C |
| Flow Gradient: | Elution began with 10% mobile phase A and 90% mobile phase B, followed by a linear gradient to 100% mobile phase A over 10 minutes. The gradient was then returned to 10% mobile phase A over 2 minutes and held for 13 minutes to equilibrate the column. MS acquisition stopped at 16 minutes. |
| Flow Rate: | 400 µL/min |
| Internal Standard: | Inosine-15N4,Thymine-d4, Glycocholate-d4, CIL NLM-4264-0.01, DLM-2742-0, DLM-1089-1 respectively |
| Solvent A: | 100% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide |
| Solvent B: | 75% acetonitrile/25% methanol; 10 mM ammonium hydroxide |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH005154 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) |
| Column Temperature: | 45°C |
| Flow Gradient: | The column was eluted isocratically at 450 μL/min with 20% mobile phase A for 3 minutes. A linear gradient was then applied to reach 100% mobile phase B over 12 minutes, followed by a 4.5-minute hold at 100% mobile phase B. The column was re-equilibrated with 20% mobile phase A for 7 minutes. MS acquisition stopped at 20 minutes. |
| Flow Rate: | 450 µL/min |
| Internal Standard: | 15R-15-methyl ProstaglandinA2,15S-15-methyl ProstaglandinE1, 15S-15-methyl ProstaglandinE2 (Cayman 10270, 13730, 14730 respectively) |
| Solvent A: | 100% Water; 0.01% Formic acid |
| Solvent B: | 100% Acetonitrile; 0.01% Acetic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS006482 |
| Analysis ID: | AN006783 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
| Ion Mode: | POSITIVE |
| MS ID: | MS006483 |
| Analysis ID: | AN006784 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | IDX |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
| Ion Mode: | POSITIVE |
| MS ID: | MS006484 |
| Analysis ID: | AN006785 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS006485 |
| Analysis ID: | AN006786 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | IDX |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
| Ion Mode: | NEGATIVE |
