Summary of Study ST002360
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 PR001515. The data can be accessed directly via it's Project DOI: 10.21228/M8VQ5S 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 | ST002360 |
| Study Title | Quantitative lipidomics of TFG-deficient B cells |
| Study Summary | The autophagy-flux-promoting protein TFG (Trk-fused gene) is up-regulated during B cell differentiation into plasma cells and supports survival of CH12 B cells. We hypothesized that quantitative proteomics analysis of CH12tfgKO B cells with intact or blocked autophagy-lysosome flux (via NH4Cl) will identify mechanisms of TFG-dependent autophagy, plasma cell biology and B cell survival. Analysis of CH12WT B cells in the presence of NH4Cl will identify proteins whose presence is continuously regulated by lysosomes independent of TFG. We determined hundreds of proteins to be controlled by TFG and/or NH4Cl. Notably, NH4Cl treatment alone increased the abundance of a cluster of cytosolic and mitochondrial translational proteins while it also reduced a number of proteins. Within the B cell relevant protein pool, BCL10 was reduced, while JCHAIN was increased in CH12tfgKO B cells. Furthermore, TFG regulated the abundance of transcription factors, such as JUNB, metabolic enzymes, such as the short-chain fatty acid activating enzyme ACOT9 or the glycolytic enzyme ALDOC. Gene ontology enrichment analysis revealed that TFG-regulated proteins localized to mitochondria and membrane-bounded organelles. Due to these findings we performed shotgun lipidomics of glycerophospholipids, uncovering that a particular phosphatidylethanolamine (PE) species, PE 32:0, which lipidates LC3 most efficiently, was less abundant while phosphatidylglycerol (PG) was more abundant in CH12tfgKO B cells. In line with the role of PG as precursor for Cardiolipin (CL), the CL content was higher in CH12tfgKO B cells and addition of PG liposomes to B cells increased the amount of CL. We propose a role for TFG in B cell activation and plasma cell biology via regulation of proteins involved in germinal center and plasma cell development, such as BCL10 or JCHAIN, as well as in lipid homeostasis, mitochondria and metabolism. |
| Institute | University of Cologne |
| Department | Faculty of Medicine and University Hospital of Cologne, Cluster of Excellence Cellular Stress Responses in Aging-associated Diseases (CECAD) |
| Laboratory | CECAD Lipidomics/Metabolomics Facility |
| Last Name | Brodesser |
| First Name | Susanne |
| Address | Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany |
| susanne.brodesser@uk-koeln.de | |
| Phone | +49 221 478 84015 |
| Submit Date | 2022-11-21 |
| Num Groups | 2 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | MS(Dir. Inf.) |
| Release Date | 2022-12-15 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001515 |
| Project DOI: | doi: 10.21228/M8VQ5S |
| Project Title: | Quantitative proteomics and lipidomics of TFG-deficient B cells provide insights into mechanisms of autophagic flux and plasma cell biology |
| Project Summary: | The autophagy-flux-promoting protein TFG (Trk-fused gene) is up-regulated during B cell differentiation into plasma cells and supports survival of CH12 B cells. We hypothesized that quantitative proteomics analysis of CH12tfgKO B cells with intact or blocked autophagy-lysosome flux (via NH4Cl) will identify mechanisms of TFG-dependent autophagy, plasma cell biology and B cell survival. Analysis of CH12WT B cells in the presence of NH4Cl will identify proteins whose presence is continuously regulated by lysosomes independent of TFG. We determined hundreds of proteins to be controlled by TFG and/or NH4Cl. Notably, NH4Cl treatment alone increased the abundance of a cluster of cytosolic and mitochondrial translational proteins while it also reduced a number of proteins. Within the B cell relevant protein pool, BCL10 was reduced, while JCHAIN was increased in CH12tfgKO B cells. Furthermore, TFG regulated the abundance of transcription factors, such as JUNB, metabolic enzymes, such as the short-chain fatty acid activating enzyme ACOT9 or the glycolytic enzyme ALDOC. Gene ontology enrichment analysis revealed that TFG-regulated proteins localized to mitochondria and membrane-bounded organelles. Due to these findings we performed shotgun lipidomics of glycerophospholipids, uncovering that a particular phosphatidylethanolamine (PE) species, PE 32:0, which lipidates LC3 most efficiently, was less abundant while phosphatidylglycerol (PG) was more abundant in CH12tfgKO B cells. In line with the role of PG as precursor for Cardiolipin (CL), the CL content was higher in CH12tfgKO B cells and addition of PG liposomes to B cells increased the amount of CL. We propose a role for TFG in B cell activation and plasma cell biology via regulation of proteins involved in germinal center and plasma cell development, such as BCL10 or JCHAIN, as well as in lipid homeostasis, mitochondria and metabolism. |
| Institute: | Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg |
| Department: | Division of Molecular Immunology, Department of Internal Medicine 3, Nikolaus-Fiebiger-Zentrum |
| Last Name: | Mielenz |
| First Name: | Dirk |
| Address: | Glückstr. 6, 91054 Erlangen, Germany |
| Email: | dirk.mielenz@fau.de |
| Phone: | +49 9131 85 39105 |
| Contributors: | Tobit D. Steinmetz, Lena Reimann, Sebastian R. Schulz, Sophia Urbanczyk, Jana Thomas, Ann-Kathrin Himmelreich, Florian Golombek, Kathrin Castiglione, Susanne Brodesser, Bettina Warscheid, Dirk Mielenz |
Subject:
| Subject ID: | SU002449 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA237000 | S11_B.cells_tfgKO_3D1 | tfgKO |
| SA237001 | S10_B.cells_tfgKO_3C6 | tfgKO |
| SA237002 | S12_B.cells_tfgKO_3G7 | tfgKO |
| SA237003 | S13_B.cells_tfgKO_1A7 | tfgKO |
| SA237004 | S14_B.cells_tfgKO_1E10 | tfgKO |
| SA237005 | S09_B.cells_tfgKO_2D2 | tfgKO |
| SA237006 | S08_B.cells_tfgKO_1C11 | tfgKO |
| SA237007 | S03_B.cells_tfgWT_4A1 | tfgWT |
| SA237008 | S02_B.cells_tfgWT_3A4 | tfgWT |
| SA237009 | S04_B.cells_tfgWT_4A5 | tfgWT |
| SA237010 | S05_B.cells_tfgWT_4C4 | tfgWT |
| SA237011 | S07_B.cells_tfgWT_4C1 | tfgWT |
| SA237012 | S06_B.cells_tfgWT_2H2 | tfgWT |
| SA237013 | S01_B.cells_tfgWT_1B2 | tfgWT |
| Showing results 1 to 14 of 14 |
Collection:
| Collection ID: | CO002442 |
| Collection Summary: | CH12tfgWT and CH12tfgKO B cells (Steinmetz et al. 2021, Autophagy 17(9):2238-2256) were cultured in R10 medium (RPMI1640 [Gibco Life Technologies, 31870-025], 10% FCS, 2 mM glutamate [Gibco Life Technologies, 25030-024], 1 mM sodium pyruvate [Gibco Life Technologies, 11360-039], 50 U/ml penicillin G, 50 μg/ml streptomycin [Gibco Life Technologies, 15140-122], 50 μM β-mercaptoethanol [Gibco Life Technologies, 31350-010]) at 37°C and 5% CO2. |
| Sample Type: | CH12 B lymphoma cells |
Treatment:
| Treatment ID: | TR002461 |
| Treatment Summary: | The cells have not undergone any further treatment. |
Sample Preparation:
| Sampleprep ID: | SP002455 |
| Sampleprep Summary: | Glycerophospholipids (PC, PE, PI, PS, PG, PA) in B cells were analyzed by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI-MS/MS) with direct infusion of the lipid extract (Shotgun Lipidomics): 14 to 45 million cells were homogenized in 300 µl of Milli-Q water using the Precellys 24 Homogenisator (Peqlab, Erlangen, Germany) at 6.500 rpm for 30 sec. The protein content of the homogenate was routinely determined using bicinchoninic acid. To 100 µl of the homogenate 400 µl of Milli-Q water, 1.875 ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PC 17:0-20:4, 132 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 131 pmol PS 17:0-20:4, 62 pmol PG 17:0/20:4, 75 pmol PA 17:0/20:4; Avanti Polar Lipids) were added. Lipids were extracted using the “One-Step Extraction” as described in Özbalci et al. 2013, Methods Mol Biol 1033:3-20. Dried lipid extracts were dissolved in 300 μl of methanol. 20 μl of the lipid extract in methanol were loaded into 96-well plates and diluted with 20 μl of 20 mM ammonium acetate in methanol. |
Combined analysis:
| Analysis ID | AN003854 |
|---|---|
| Analysis type | MS |
| Chromatography type | None (Direct infusion) |
| Chromatography system | none |
| Column | none |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 6500 QTrap |
| Ion Mode | POSITIVE |
| Units | counts per second (cps) |
Chromatography:
| Chromatography ID: | CH002852 |
| Chromatography Summary: | Lipid infusion and ionization was conducted using Nano-ESI chips with the TriVersa NanoMate operated by the ChipSoft Software (Advion) under the following settings: sample infusion volume: 14 μl, volume of air to aspirate after sample: 1 μl, air gap before chip: enabled, aspiration delay: 0 s, prepiercing: with mandrel, spray sensing: enabled, cooling temperature: 14°C, gas pressure: 0.5 psi, ionization voltage: 1.4 kV, and vent headspace: enabled. Prewetting was done once. |
| Instrument Name: | none |
| Column Name: | none |
| Chromatography Type: | None (Direct infusion) |
MS:
| MS ID: | MS003595 |
| Analysis ID: | AN003854 |
| Instrument Name: | ABI Sciex 6500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Mass spectrometric analysis was performed using the QTRAP 6500 (SCIEX) operated by Analyst 1.6.3. The following instrument dependent settings were used: curtain gas, 20 psi; CAD gas, medium; and interface heater temperature, 100°C. PC analysis was performed in the positive ion mode by scanning for precursors of m/z 184 at a collision energy of 35 eV. PE, PS, PG, PI, and PA measurements were performed in the positive ion mode by scanning for neutral losses of 141, 185, 189, 277, and 115 D at CE of 25 eV. The value for the declustering potential was 100 V (Özbalci et al. 2013, Methods Mol Biol 1033:3-20). Scanning was performed in a mass range of m/z 650–900 D and at a scan rate of 200 D/s. 61 MCA spectra were accumulated. Mass spectra were processed by the LipidView Software Version 1.2 (SCIEX) for identification and quantification of glycerophospholipids. Endogenous glycerophospolipids were quantified by referring their peak areas to those of the internal standards. |
| Ion Mode: | POSITIVE |
