Summary of Study ST002140

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 PR001355. The data can be accessed directly via it's Project DOI: 10.21228/M8K70K 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	ST002140
Study Title	Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling flux of the TCA cycle
Study Summary	The function of mitochondrial respiration during B cell fate decisions and differentiation 55 remained equivocal. This study reveals that selection for mitochondrial fitness occurs during B 56 cell activation and is essential for subsequent plasma cell differentiation. By expressing a 57 mutated mitochondrial helicase in transitional B cells, we depleted mitochondrial DNA during 58 B cell maturation, resulting in reduced oxidative phosphorylation. Although no changes in 59 follicular B cell development were evident, germinal centers, class switch recombination to 60 IgG, plasma cell maturation and humoral immunity were diminished. Defective oxidative 61 phosphorylation led to aberrant flux of the tricarboxylic acid cycle and lowered the amount of 62 saturated phosphatidic acid. Consequently, mTOR activity and BLIMP1 induction were 63 curtailed whereas HIF1 _and glycolysis were amplified. Exogenous phosphatidic acid 64 increased mTOR activity in activated B cells. Hence, mitochondrial function is required and 65 selected for in activated B cells for the successful generation of functional plasma cells.
Institute	University of Erlangen-Nürnberg
Department	Division of Molecular Immunology.Universitätsklinikum Erlangen, Nikolaus Fibinger Zentrum
Laboratory	Prof. Mielenz
Last Name	Mielenz
First Name	Dirk
Address	Nikolaus-Fiebiger-Zentrum, Glückstraße 6, 91054 Erlangen
Email	dirk.mielenz@fau.de
Phone	++49 9131 8539105
Submit Date	2022-04-06
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS/MS(Dir. Inf.)
Release Date	2022-05-02
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP002231
Sampleprep Summary:	Glycerophospholipid analysis Glycerophospholipids (PCH, 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 x 106 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, 1.875ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PCH 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. Lipid extraction and Nano-ESI-MS/MS analysis were performed as previously described (Kumar et al., 2015). Endogenous glycerophospolipids were quantified by referring their peak areas to those of the internal standards. The calculated glycerophospolipid amounts were normalized to the protein content of the tissue homogenate. Metabolomics of phosphorylated metabolites und carbonic acids Experimental Setup I: Splenic B cells were isolated, activated with LPS and viable cells, only GFP+ for DNT, were sorted after 3 days using flow cytometry. Perchloric acid extraction and metabolic profiling was performed as previously published measured by LCMS/MS on an QTrap 3200 (Sciex) (Hofmann et al., 2011). Ref.: Kumar, V., Bouameur, J.E., Bar, J., Rice, R.H., Hornig-Do, H.T., Roop, D.R., Schwarz, N., Brodesser, 1120 S., Thiering, S., Leube, R.E., et al. (2015). A keratin scaffold regulates epidermal barrier 1121 formation, mitochondrial lipid composition, and activity. J Cell Biol 211, 1057–1075. Ref.: Hofmann, J., Bornke, F., Schmiedl, A., Kleine, T., and Sonnewald, U. (2011). Detecting functional groups of Arabidopsis mutants by metabolic profiling and evaluation of pleiotropic responses. 10Front Plant Sci 2, 82.

Sampleprep ID:

SP002231

Sampleprep Summary:

Glycerophospholipid analysis Glycerophospholipids (PCH, 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 x 106 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, 1.875ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PCH 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. Lipid extraction and Nano-ESI-MS/MS analysis were performed as previously described (Kumar et al., 2015). Endogenous glycerophospolipids were quantified by referring their peak areas to those of the internal standards. The calculated glycerophospolipid amounts were normalized to the protein content of the tissue homogenate. Metabolomics of phosphorylated metabolites und carbonic acids Experimental Setup I: Splenic B cells were isolated, activated with LPS and viable cells, only GFP+ for DNT, were sorted after 3 days using flow cytometry. Perchloric acid extraction and metabolic profiling was performed as previously published measured by LCMS/MS on an QTrap 3200 (Sciex) (Hofmann et al., 2011). Ref.: Kumar, V., Bouameur, J.E., Bar, J., Rice, R.H., Hornig-Do, H.T., Roop, D.R., Schwarz, N., Brodesser, 1120 S., Thiering, S., Leube, R.E., et al. (2015). A keratin scaffold regulates epidermal barrier 1121 formation, mitochondrial lipid composition, and activity. J Cell Biol 211, 1057–1075. Ref.: Hofmann, J., Bornke, F., Schmiedl, A., Kleine, T., and Sonnewald, U. (2011). Detecting functional groups of Arabidopsis mutants by metabolic profiling and evaluation of pleiotropic responses. 10Front Plant Sci 2, 82.