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
Email	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:

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.

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.