{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002322","ANALYSIS_ID":"AN003789","VERSION":"1","CREATED_ON":"10-23-2022"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics reveals global metabolic changes between WT and SCAP deficient B cells","PROJECT_TYPE":"Purified mouse B cells, stimulated in vitro","PROJECT_SUMMARY":"Cellular metabolism regulates almost all critical cellular activities including cell growth, proliferation, energy homeostasis and signaling transduction. SCAP controls SREBP signaling, an important pathway regulating cellular lipid biosynthesis. SCAP KO B cells have multiple defects and cannot form germinal centers. To test whether these defects of SCAP KO B cells are associated with an altered global metabolic state. We stimulated B cells isolated from SCAPfl/fl CD19Cre/+ mice (KO) and SCAP+/+ CD19Cre/+ mice (WT) with ⍺-CD40 or LPS for 24 and 48 hours, followed by targeted metabolomics analysis to evaluate global metabolic changes. We found that the metabolic profiles of unstimulated SCAP KO and WT B cells were largely overlapping, corroborating our findings that SCAP deficiency did not affect B cell development or their metabolic profile at steady state. In contrast, after stimulation with either LPS or ⍺-CD40, SCAP KO and WT B cells were clearly separated at 24 hours, with an even more pronounced separation at 48 hours. SREBP signaling can regulated the metabolism of many ceramides and sphingolipids. For example, lactosyl-N-palmitoyl-sphingosine (d18:1/16:0), one of the lactosylceramides (LacCer), is highly accumulated in SCAP KO B cells activated by either ⍺-CD40 or LPS. Strikingly, besides lipid metabolism, SCAP deficiency also alters many other metabolites that belong to different metabolic pathways such as energy, amino acids, peptide, cofactors and vitamins, nucleotide, carbohydrate, and xenobiotics. Taken together, the metabolomics analysis revealed previously unrecognized roles of SREBP signaling in regulating multiple cellular activities in activated B cells.","INSTITUTE":"Indiana University","LAST_NAME":"Luo","FIRST_NAME":"Wei","ADDRESS":"950 W Walnut Street - R2 E304, Indianapolis, IN, 46202, USA","EMAIL":"wl47@iu.edu","PHONE":"3172748042","DOI":"http://dx.doi.org/10.21228/M8CT5G"},

"STUDY":{"STUDY_TITLE":"Metabolomics study comparing SCAP KO and WT B cells","STUDY_TYPE":"Purified mouse B cells, stimulated ex vivo","STUDY_SUMMARY":"Splenic B cells isolated from SCAP+/+ CD19-Cre (WT) and SCAPfl/fl CD19-Cre (KO) mice were stimulated with LPS or anti-CD40 for 24 and 48 hours. Cells were then analyzed by metabolomics. Metabolomics reveals global metabolic changes in SCAP deficient B cells.","INSTITUTE":"Indiana University School of Medicine","LAST_NAME":"Luo","FIRST_NAME":"Wei","ADDRESS":"950 W Walnut Street - R2 E304","EMAIL":"wl47@iu.edu","PHONE":"3172748042","SUBMIT_DATE":"2022-10-19"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"SCAP fl/fl CD19-Cre (KO) and SCAP+/+ CD19-Cre (WT)","CELL_STRAIN_DETAILS":"Splenic B cell"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"KO_CD40_24hr_1",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"anti-CD40","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_CD40_24hr_2",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"anti-CD40","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_CD40_24hr_3",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"anti-CD40","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_CD40_24hr_4",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"anti-CD40","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_CD40_48hr_1",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"anti-CD40","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_CD40_48hr_2",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"anti-CD40","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_CD40_48hr_3",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"anti-CD40","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_CD40_48hr_4",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"anti-CD40","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_0hr_1",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"Freshly isolated (No treatment)","TIME_POINT":"0hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_0hr_2",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"Freshly isolated (No treatment)","TIME_POINT":"0hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_0hr_3",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"Freshly isolated (No treatment)","TIME_POINT":"0hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_0hr_4",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"Freshly isolated (No treatment)","TIME_POINT":"0hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_LPS_24hr_1",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"LPS","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_LPS_24hr_2",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"LPS","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_LPS_24hr_3",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"LPS","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_LPS_24hr_4",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"LPS","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_LPS_48hr_1",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"LPS","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_LPS_48hr_2",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"LPS","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_LPS_48hr_3",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"LPS","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"KO_LPS_48hr_4",
"Factors":{"Genotype":"SCAP KO","TREATMENT":"LPS","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_CD40_24hr_1",
"Factors":{"Genotype":"WT","TREATMENT":"anti-CD40","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_CD40_24hr_2",
"Factors":{"Genotype":"WT","TREATMENT":"anti-CD40","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_CD40_24hr_3",
"Factors":{"Genotype":"WT","TREATMENT":"anti-CD40","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_CD40_24hr_4",
"Factors":{"Genotype":"WT","TREATMENT":"anti-CD40","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_CD40_48hr_1",
"Factors":{"Genotype":"WT","TREATMENT":"anti-CD40","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_CD40_48hr_2",
"Factors":{"Genotype":"WT","TREATMENT":"anti-CD40","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_CD40_48hr_3",
"Factors":{"Genotype":"WT","TREATMENT":"anti-CD40","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_CD40_48hr_4",
"Factors":{"Genotype":"WT","TREATMENT":"anti-CD40","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_0hr_1",
"Factors":{"Genotype":"WT","TREATMENT":"Freshly isolated (No treatment)","TIME_POINT":"0hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_0hr_2",
"Factors":{"Genotype":"WT","TREATMENT":"Freshly isolated (No treatment)","TIME_POINT":"0hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_0hr_3",
"Factors":{"Genotype":"WT","TREATMENT":"Freshly isolated (No treatment)","TIME_POINT":"0hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_0hr_4",
"Factors":{"Genotype":"WT","TREATMENT":"Freshly isolated (No treatment)","TIME_POINT":"0hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_LPS_24hr_1",
"Factors":{"Genotype":"WT","TREATMENT":"LPS","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_LPS_24hr_2",
"Factors":{"Genotype":"WT","TREATMENT":"LPS","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_LPS_24hr_3",
"Factors":{"Genotype":"WT","TREATMENT":"LPS","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_LPS_24hr_4",
"Factors":{"Genotype":"WT","TREATMENT":"LPS","TIME_POINT":"24hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_LPS_48hr_1",
"Factors":{"Genotype":"WT","TREATMENT":"LPS","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_LPS_48hr_2",
"Factors":{"Genotype":"WT","TREATMENT":"LPS","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_LPS_48hr_3",
"Factors":{"Genotype":"WT","TREATMENT":"LPS","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
},
{
"Subject ID":"-",
"Sample ID":"WT_LPS_48hr_4",
"Factors":{"Genotype":"WT","TREATMENT":"LPS","TIME_POINT":"48hr"},
"Additional sample data":{"Cell type":"Splenic B cells"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Mouse splenic B cells were isolated using STEM CELL B cell isolation kit (Cat:19854)","SAMPLE_TYPE":"B cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Splenic B cells isolated from SCAP+/+ CD19-Cre (WT) and SCAPfl/fl CD19-Cre (KO) mice were stimulated with 10 micro g/ml LPS or 5 micro g/ml anti-CD40 for 24 and 48 hours. Freshly isolated untreated WT and KO B cells were used as untreated control."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Sample preparation: Proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phases (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis."},

"CHROMATOGRAPHY":{"METHODS_FILENAME":"B_cell_MS_protocol.docx","INSTRUMENT_NAME":"Waters ACQUITY ultra-performance liquid chromatography (UPLC)","COLUMN_NAME":"Waters UPLC BEH C18 (2.1x100 mm, 1.7 um)/Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)","CHROMATOGRAPHY_TYPE":"Reversed phase/HILIC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","ANALYSIS_PROTOCOL_FILE":"B_cell_MS_protocol.docx"},

"MS":{"INSTRUMENT_NAME":"Thermo Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","MS_COMMENTS":"Analysis was performed by Metabolon. Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS): All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible with each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions; however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slightly between methods but covered 70-1000 m/z.","ION_MODE":"UNSPECIFIED"},

"MS_METABOLITE_DATA":{
"Units":"Normalized AUC",

"Data":[{"Metabolite":"1-(1-enyl-oleoyl)-2-oleoyl-GPE (P-18:1/18:1)*","KO_CD40_24hr_1":"0.5936","KO_CD40_24hr_2":"0.8552","KO_CD40_24hr_3":"0.5392","KO_CD40_24hr_4":"0.6289","KO_CD40_48hr_1":"0.9883","KO_CD40_48hr_2":"1.5664","KO_CD40_48hr_3":"1.4518","KO_CD40_48hr_4":"1.6833","KO_0hr_1":"0.9634","KO_0hr_2":"0.5815","KO_0hr_3":"0.8983","KO_0hr_4":"0.6128","KO_LPS_24hr_1":"0.7771","KO_LPS_24hr_2":"0.6663","KO_LPS_24hr_3":"1.0000","KO_LPS_24hr_4":"0.8729","KO_LPS_48hr_1":"4.1740","KO_LPS_48hr_2":"3.3314","KO_LPS_48hr_3":"2.4405","KO_LPS_48hr_4":"3.5876","WT_CD40_24hr_1":"0.8746","WT_CD40_24hr_2":"0.6322","WT_CD40_24hr_3":"0.4604","WT_CD40_24hr_4":"0.7069","WT_CD40_48hr_1":"2.4631","WT_CD40_48hr_2":"2.3721","WT_CD40_48hr_3":"1.9158","WT_CD40_48hr_4":"1.9255","WT_0hr_1":"0.3751","WT_0hr_2":"0.8836","WT_0hr_3":"0.5502","WT_0hr_4":"1.1727","WT_LPS_24hr_1":"1.3349","WT_LPS_24hr_2":"1.0626","WT_LPS_24hr_3":"1.2028","WT_LPS_24hr_4":"1.0061","WT_LPS_48hr_1":"1.2838","WT_LPS_48hr_2":"3.0478","WT_LPS_48hr_3":"4.2237","WT_LPS_48hr_4":"2.6341"},{"Metabolite":"1-(1-enyl-oleoyl)-GPE (P-18:1)*","KO_CD40_24hr_1":"0.2028","KO_CD40_24hr_2":"0.2028","KO_CD40_24hr_3":"0.2028","KO_CD40_24hr_4":"0.2028","KO_CD40_48hr_1":"0.3283","KO_CD40_48hr_2":"0.5112","KO_CD40_48hr_3":"0.5411","KO_CD40_48hr_4":"0.3536","KO_0hr_1":"0.2028","KO_0hr_2":"0.2028","KO_0hr_3":"0.2028","KO_0hr_4":"0.2028","KO_LPS_24hr_1":"0.6673","KO_LPS_24hr_2":"0.6118","KO_LPS_24hr_3":"1.4455","KO_LPS_24hr_4":"0.9404","KO_LPS_48hr_1":"1.4517","KO_LPS_48hr_2":"0.9735","KO_LPS_48hr_3":"0.9175","KO_LPS_48hr_4":"1.1064","WT_CD40_24hr_1":"0.5328","WT_CD40_24hr_2":"0.5127","WT_CD40_24hr_3":"0.2028","WT_CD40_24hr_4":"0.3396","WT_CD40_48hr_1":"1.7243","WT_CD40_48hr_2":"1.9255","WT_CD40_48hr_3":"1.3247","WT_CD40_48hr_4":"1.4099","WT_0hr_1":"0.2028","WT_0hr_2":"0.2028","WT_0hr_3":"0.2028","WT_0hr_4":"0.2028","WT_LPS_24hr_1":"1.6992","WT_LPS_24hr_2":"1.6449","WT_LPS_24hr_3":"1.3988","WT_LPS_24hr_4":"1.0265","WT_LPS_48hr_1":"0.5362","WT_LPS_48hr_2":"1.4917","WT_LPS_48hr_3":"1.9763","WT_LPS_48hr_4":"1.2163"},{"Metabolite":"1-(1-enyl-palmitoyl)-2-arachidonoyl-GPE (P-16:0/20:4)*","KO_CD40_24hr_1":"0.9231","KO_CD40_24hr_2":"1.2488","KO_CD40_24hr_3":"0.8103","KO_CD40_24hr_4":"0.9530","KO_CD40_48hr_1":"1.1960","KO_CD40_48hr_2":"1.6831","KO_CD40_48hr_3":"1.5526","KO_CD40_48hr_4":"1.6943","KO_0hr_1":"1.4505","KO_0hr_2":"0.8222","KO_0hr_3":"1.1303","KO_0hr_4":"0.9308","KO_LPS_24hr_1":"0.7912","KO_LPS_24hr_2":"0.7102","KO_LPS_24hr_3":"1.1616","KO_LPS_24hr_4":"1.0511","KO_LPS_48hr_1":"2.1241","KO_LPS_48hr_2":"2.4031","KO_LPS_48hr_3":"1.5673","KO_LPS_48hr_4":"2.1883","WT_CD40_24hr_1":"1.0843","WT_CD40_24hr_2":"0.7973","WT_CD40_24hr_3":"1.1822","WT_CD40_24hr_4":"0.8670","WT_CD40_48hr_1":"1.2310","WT_CD40_48hr_2":"1.1511","WT_CD40_48hr_3":"1.1178","WT_CD40_48hr_4":"1.0377","WT_0hr_1":"0.9488","WT_0hr_2":"1.2927","WT_0hr_3":"0.9654","WT_0hr_4":"1.7819","WT_LPS_24hr_1":"1.0138","WT_LPS_24hr_2":"0.8042","WT_LPS_24hr_3":"0.9135","WT_LPS_24hr_4":"0.8079","WT_LPS_48hr_1":"0.3860","WT_LPS_48hr_2":"0.9848","WT_LPS_48hr_3":"1.2257","WT_LPS_48hr_4":"0.9318"},{"Metabolite":"1-(1-enyl-palmitoyl)-2-linoleoyl-GPE 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