Metabolite identification was done with MS-DIAL version 4.70 in lipidomics mode. Default lipid library was used (Msp20210527163602_converted.lbm2).
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - reverse-phase","Liquid Chromatography MS - positive - reverse-phase"],"chromatography_protocol":["Chromatographic separation was performed using a Vanquish UHPLC system (Thermo Scientific), with a reverse-phase Acquity BEH C18 (1.7 µm, 2.1×150mm, Waters) column at a flow rate of 250 ul/min. Mobile phases were made up of 10 mM ammonium formate and 0.1% formic acid in (A) H2O/CH3CN (40:60) and in (B) CH3CN/ iPrOH (10:90). Gradient conditions were as follows: 0–1 min, B = 20 %; 1–8 min, B = 20– 100 %; 8–10 min, B = 100 %; 10–10.5 min, B = 100–20 %; 10.5–15 min, B = 20%. The total chromatographic run time was 15 min.
"],"publication":["B cell expression of the enzyme PexRAP, an intermediary in ether lipid biosynthesis, promotes antibody responses and germinal center size. 10.1101/2024.10.17.618760. PMID:39464149"],"submitter_affiliation":["Vanderbilt","Vanderbilt University & Medical Center","Vanderbilt University"],"submitter_name":["Mark Boothby","CSV fourkin","Sergei Chetyrkin"],"organism_part":["B-lymphocyte cell line","mixture","solvent","buffer"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["Lipid extraction
To prepare samples, a one-phase method was used to extract lipids[1]. Briefly, 0.5 mL of MeOH/MTBE/CHCl3 mix (1.3:1:1) was added to a frozen pellet of B-cells (1×10^6 cells total), spiked with 10 uL EquiSPLASH-lipidomics internal standard mix (Avanti Research), briefly vortexed and shaken gently for 20 min, followed by centrifugation at 20,000 x g for 15 min at 10°C. The supernatant was transferred to a clean Eppendorf tube, evaporated under a gentle stream of N2 gas, and resuspended in 100 uL methanol/CHCl3 (9:1) and 2uL were used for LC-HRMS (high-resolution MS) analysis.
Ref:
[1] Pellegrino, R.M., Di Veroli, A., Valeri, A., Goracci, L. and Cruciani, G., 2014. LC/MS lipid profiling from human serum: a new method for global lipid extraction. Analytical and bioanalytical chemistry, 406(30), pp.7937-7948.
"],"organism":["blank sample","Mus musculus","solvent blank"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS13880"],"author":["SCV fourkin. Vanderbilt University. fourkin@gmail.com.","Sergiy Chetyrkin. Universidad Univer. Mass Spectrometry Research Center, MRB-III 9th floor. sergei.chetyrkin@vanderbilt.edu.","Mark Boothby. Vanderbilt University Medical Center. mark.boothby@vumc.org."],"data_transformation_protocol":["High resolution mass spectrometry data were processed with MS-DIAL version 4.70 in lipidomics mode (86). MS1, and MS2 tolerances were set to 0.01 and 0.025 Da respectively. Minimum peak height was set to 30,000 to decrease the number of false positive hits. Peaks were aligned on a quality control (QC) reference file with RT tolerance of 0.1 min and mass tolerance of 0.015 Da. Default lipid library was used (Msp20210527163602_converted.lbm2), solvent type was set to HCOONH4 to match the solvent used for separation, and the identification score cut off was set to 80%. All lipid classes were made available for the search.
After lipid identification was completed, MS-DIAL results were exported into Excel and cleaned using minimum RSD for QC samples set to 20% and minimum ratio of QC to Blank set to 10. For species identified in both PexRAP-depleted (Dhrs7b Δ/Δ) B cells and controls, mean levels (areas under peak curves) and their variance were analyzed in Excel.
"],"study_factor":["Experimental group","Differentiation state"],"submitter_email":["mark.boothby@vumc.org","sergei.chetyrkin@vanderbilt.edu","fourkin@gmail.com"],"sample_collection_protocol":["After tamoxifen administration to mice (CreERT2 or CreERT2, Dhrs7b f/f), or for C57Bl/6 mice not treated with tamoxifen, pools of B lymphocytes (B cells) were generated by using negative selection on single cell suspensions of splenocytes, as in the Methods of the paper linked to this data deposition. B cells pools either were snap-frozen ('naive' / unactivated samples) or subjected to in vitro activation with anti-CD40 mAb and culture (48 hr) in BAFF (as detailed in the Methods of PMID: 39464149), followed by snap freezing on dry ice.
Frozen pellet of B-cells (1x106 cells total), spiked with 10 uL EquiSPLASH-lipidomics internal standard mix (Avanti Research). Along with samples, there were three different blanks used to control various aspects of sample preparation: 1) AB - this plank was PBS buffer (same buffer I used to wash cells) processed along with samples (extracted, dried, reconstituted); 2) AIS - this blank was PBS buffer spiked with internal standards and processed along with samples (extracted, dried, reconstituted); 3) solvent blank - reconstitution solvent, not processed.
Each sample was injected 2 times - 1 injection in positive ESI mode followed by 1 in negative mode. Pooled QCs were injected to assess the performance of the LC and MS instruments at the beginning, in the middle and at the end of each sequence.
"],"omics_type":["Metabolomics"],"study_design":["B cell","imaging mass spectrometry","Mouse","Lipidomics"],"curator_keywords":["B cell","imaging mass spectrometry","Mouse","Lipidomics"],"mass_spectrometry_protocol":["Mass spectra were acquired using a Q Exactive HF (Thermo Fisher Scientific) quadrupole/orbitrap mass spectrometer, over a precursor ion scan range of m/z 200 to 1600 at a resolving power of 60,000 using the following HESI-II source parameters: spray voltage 4 kV (3 kV in negative mode); capillary temperature 250 °C; S-lens RF level 60 V; N2 sheath gas 40; N2 auxiliary gas 10; auxiliary gas temperature 350 °C. MS/MS spectra were acquired for the top-seven most abundant precursor ions with an MS/MS AGC target of 1e5, a maximum MS/MS injection time of 100 ms, and a normalized collision energy of 15, 30, 40.
"],"pubmed_abstract":["The qualities of antibody (Ab) responses provided by B lymphocytes and their plasma cell (PC) descendants are crucial facets of responses to vaccines and microbes. Metabolic processes and products regulate aspects of B cell proliferation and differentiation into germinal center (GC) and PC states as well as Ab diversification. However, there is little information about lymphoid cell-intrinsic functions of enzymes that mediate ether lipid biosynthesis, including a major class of membrane phospholipids. Imaging mass spectrometry (IMS) results had indicated that concentrations of a number of these phospholipids were substantially enhanced in GC compared to the background average in spleens. However, it was not clear if biosynthesis in B cells was a basis for this finding, or whether such cell-intrinsic biosynthesis contributes to B cell physiology or Ab responses. Ether lipid biosynthesis can involve the enzyme PexRAP, the product of the Dhrs7b gene. Using combinations of IMS and immunization experiments in mouse models with inducible Dhrs7b loss-of-function, we now show that B lineage-intrinsic expression of PexRAP promotes the magnitude and affinity maturation of a serological response. Moreover, the data revealed a Dhrs7b -dependent increase in ether phospholipids in primary follicles with a more prominent increase in GC. Mechanistically, PexRAP impacted B cell proliferation via enhanced survival associated with controlling levels of ROS and membrane peroxidation. These findings reveal a vital role of this peroxisomal enzyme in B cell homeostasis and the physiology of humoral immunity."],"pubmed_title":["B cell expression of the enzyme PexRAP, an intermediary in ether lipid biosynthesis, promotes antibody responses and germinal center size."],"pubmed_authors":["Cho Sung Hoon SH, Jones Marissa A MA, Meyer Kaylor K, Anderson David M DM, Chetyrkin Sergei S, Calcutt M Wade MW, Caprioli Richard M RM, Semenkovich Clay F CF, Boothby Mark R MR"],"additional_accession":[]},"is_claimable":false,"name":"B cell expression of the enzyme PexRAP, an intermediary in ether lipid biosynthesis, promotes antibody responses and germinal center size.","description":"The qualities of antibody (Ab) responses provided by B lymphocytes and their plasma cell (PC) descendants are crucial facets of responses to vaccines and microbes. Metabolic processes and products regulate aspects of B cell proliferation and differentiation into germinal center (GC) and PC states as well as Ab diversification. However, there is little information about lymphoid cell-intrinsic functions of enzymes that mediate ether lipid biosynthesis, including a major class of membrane phospholipids. Imaging mass spectrometry (IMS) results had indicated that concentrations of a number of these phospholipids were substantially enhanced in GC compared to the background average in spleens. However, it was not clear if biosynthesis in B cells was a basis for this finding, or whether such cell-intrinsic biosynthesis contributes to B cell physiology or Ab responses. Ether lipid biosynthesis can involve the enzyme PexRAP, the product of the Dhrs7b gene. Using combinations of IMS and immunization experiments in mouse models with inducible Dhrs7b loss-of-function, we now show that B lineage-intrinsic expression of PexRAP promotes the magnitude and affinity maturation of a serological response. Moreover, the data revealed a Dhrs7b-dependent increase in ether phospholipids in primary follicles with a more prominent increase in GC. Mechanistically, PexRAP impacted B cell proliferation via enhanced survival associated with controlling levels of ROS and membrane peroxidation. These findings reveal a vital role of this peroxisomal enzyme in B cell homeostasis and the physiology of humoral immunity.
","dates":{"publication":"2026-04-13","submission":"2026-02-10"},"accession":"MTBLS13880","cross_references":{"pubmed":["39464149"]}}