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.

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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.

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Study IDST002360
Study TitleQuantitative lipidomics of TFG-deficient B cells
Study SummaryThe 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
DepartmentFaculty of Medicine and University Hospital of Cologne, Cluster of Excellence Cellular Stress Responses in Aging-associated Diseases (CECAD)
LaboratoryCECAD Lipidomics/Metabolomics Facility
Last NameBrodesser
First NameSusanne
AddressJoseph-Stelzmann-Str. 26, 50931 Cologne, Germany
Emailsusanne.brodesser@uk-koeln.de
Phone+49 221 478 84015
Submit Date2022-11-21
Num Groups2
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailMS(Dir. Inf.)
Release Date2022-12-15
Release Version1
Susanne Brodesser Susanne Brodesser
https://dx.doi.org/10.21228/M8VQ5S
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype
SA237000S11_B.cells_tfgKO_3D1tfgKO
SA237001S10_B.cells_tfgKO_3C6tfgKO
SA237002S12_B.cells_tfgKO_3G7tfgKO
SA237003S13_B.cells_tfgKO_1A7tfgKO
SA237004S14_B.cells_tfgKO_1E10tfgKO
SA237005S09_B.cells_tfgKO_2D2tfgKO
SA237006S08_B.cells_tfgKO_1C11tfgKO
SA237007S03_B.cells_tfgWT_4A1tfgWT
SA237008S02_B.cells_tfgWT_3A4tfgWT
SA237009S04_B.cells_tfgWT_4A5tfgWT
SA237010S05_B.cells_tfgWT_4C4tfgWT
SA237011S07_B.cells_tfgWT_4C1tfgWT
SA237012S06_B.cells_tfgWT_2H2tfgWT
SA237013S01_B.cells_tfgWT_1B2tfgWT
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 QTRAP
MS instrument name SCIEX QTRAP 6500
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:SCIEX QTRAP 6500
Instrument Type:QTRAP
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
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