{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000560","ANALYSIS_ID":"AN000860","VERSION":"1","CREATED_ON":"02-08-2024"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics of immunoglobulin-producing cells in IgA nephropathy","PROJECT_SUMMARY":"IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by deposits of IgA-containing immune complexes in the kidney glomeruli, as first described by Berger and Hinglais in 1968. IgAN is a major cause of end-stage renal disease with its associated cardio-renal morbidity and mortality. Analyses of the IgA deposits revealed that the IgA is exclusively of the IgA1 subclass and that this IgA1 is aberrantly glycosylated, deficient in galactose in some O-glycans (Gd-IgA1). Patients with IgAN have elevated serum levels of Gd-IgA1 bound by anti-glycan autoantibodies in circulating immune complexes (CIC) that are fundamental in driving disease pathology in an autoimmune process. We have recently shown that elevated serum levels of Gd-IgA1 in patients with IgAN predict disease progression. Thus, understanding the mechanisms behind Gd-IgA1 production will improve future treatment options, as there is presently no disease-specific therapy. A total of 24 cell pellets (4 replicates from 6 cell lines) were analyzed by LCMS metabolomics. Immortalized immunoglobulin-producing cell lines were generated from peripheral-blood lymphocytes from patients with IgAN and healthy controls as described in Suzuki, H., Moldoveanu, Z., Hall, S., et al. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Invest. 2008, 118, 629-639.","INSTITUTE":"University of Alabama, Birmingham","DEPARTMENT":"Departments of Microbiology and Medicine","LAST_NAME":"Novak","FIRST_NAME":"Jan","ADDRESS":"845 19th St.South, BBRB 761A","EMAIL":"jannovak@uab.edu","PHONE":"205-934-4480","FUNDING_SOURCE":"NIH/NIGMS Grant # K01GM109320 to Jessica Gooding; NIDDK Grant # K01DK106341 to Colin Reily; NIDDK Grant # DK078244 to Jan Novak and NIH Common Fund ERCMRC Grant # U24DK097193 to Susan Sumner","DOI":"http://dx.doi.org/10.21228/M8XG7R","CONTRIBUTORS":"Jessica Gooding1,2, Colin Reily3, Courtney Whitaker1,2, Hieu Sy Vu1,2, Zach Acuff1,2, Susan McRitchie1,2, Bruce A. Julian3, Jan Novak3, Susan Sumner2,4 1Analytical Chemistry & Pharmaceutics, RTI International, RTP, NC 2NIH Eastern Regional Comprehensive Metabolomics Resource Core (ERCMRC) at UNC Chapel Hill, NC 3Departments of Microbiology and Medicine, University of Alabama at Birmingham, Birmingham, AL 4Nutrition Research Institute, University of North Carolina, Chapel Hill, NC"},

"STUDY":{"STUDY_TITLE":"Metabolomics of immunoglobulin-producing cells in IgA nephropathy","STUDY_SUMMARY":"IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by deposits of IgA-containing immune complexes in the kidney glomeruli, as first described by Berger and Hinglais in 1968. IgAN is a major cause of end-stage renal disease with its associated cardio-renal morbidity and mortality. Analyses of the IgA deposits revealed that the IgA is exclusively of the IgA1 subclass and that this IgA1 is aberrantly glycosylated, deficient in galactose in some O-glycans (Gd-IgA1). Patients with IgAN have elevated serum levels of Gd-IgA1 bound by anti-glycan autoantibodies in circulating immune complexes (CIC) that are fundamental in driving disease pathology in an autoimmune process. We have recently shown that elevated serum levels of Gd-IgA1 in patients with IgAN predict disease progression. Thus, understanding the mechanisms behind Gd-IgA1 production will improve future treatment options, as there is presently no disease-specific therapy. A total of 24 cell pellets (4 replicates from 6 cell lines) were analyzed by LCMS metabolomics. Immortalized immunoglobulin-producing cell lines were generated from peripheral-blood lymphocytes from patients with IgAN and healthy controls as described in Suzuki, H., Moldoveanu, Z., Hall, S., et al. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Invest. 2008, 118, 629-639.","INSTITUTE":"RTI International","LABORATORY":"NIH Eastern Regional Comphrehensive Metabolomics Resource Core at UNC Chapel Hill (ERCMRC)","LAST_NAME":"Sumner","FIRST_NAME":"Susan","ADDRESS":"3040 E. Cornwallis Road, Research Triangle Park, NC 27709","EMAIL":"susan_sumner@unc.edu","PHONE":"704-250-5000","SUBMIT_DATE":"2017-02-17"},

"SUBJECT":{"SUBJECT_TYPE":"Cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_STRAIN_DETAILS":"Suzuki, H., Moldoveanu, Z., Hall, S., et al. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Inv. 2008, 118, 629-639","CELL_PRIMARY_IMMORTALIZED":"immortalized","CELL_COUNTS":"1x10^7 cells / pellet","SPECIES_GROUP":"Human"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"HC1_1",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC1","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC1_1 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC1_1 _NEG.raw","Replicate":"1"}
},
{
"Subject ID":"-",
"Sample ID":"HC1_2",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC1","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC1_2 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC1_2 _NEG.raw","Replicate":"2"}
},
{
"Subject ID":"-",
"Sample ID":"HC1_3",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC1","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC1_3 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC1_3 _NEG.raw","Replicate":"3"}
},
{
"Subject ID":"-",
"Sample ID":"HC1_4",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC1","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC1_4 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC1_4 _NEG.raw","Replicate":"4"}
},
{
"Subject ID":"-",
"Sample ID":"HC2_1",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC2","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC2_1 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC2_1 _NEG.raw","Replicate":"1"}
},
{
"Subject ID":"-",
"Sample ID":"HC2_2",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC2","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC2_2 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC2_2 _NEG.raw","Replicate":"2"}
},
{
"Subject ID":"-",
"Sample ID":"HC2_3",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC2","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC2_3 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC2_3 _NEG.raw","Replicate":"3"}
},
{
"Subject ID":"-",
"Sample ID":"HC2_4",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC2","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC2_4 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC2_4 _NEG.raw","Replicate":"4"}
},
{
"Subject ID":"-",
"Sample ID":"HC3_1",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC3","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC3_1 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC3_1 _NEG.raw","Replicate":"1"}
},
{
"Subject ID":"-",
"Sample ID":"HC3_2",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC3","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC3_2 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC3_2 _NEG.raw","Replicate":"2"}
},
{
"Subject ID":"-",
"Sample ID":"HC3_3",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC3","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC3_3 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC3_3 _NEG.raw","Replicate":"3"}
},
{
"Subject ID":"-",
"Sample ID":"HC3_4",
"Factors":{"Phenotype":"HC"},
"Additional sample data":{"Cell Line ID":"HC3","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_HC3_4 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_HC3_4 _NEG.raw","Replicate":"4"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN1_1",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN1","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN1_1 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN1_1 _NEG.raw","Replicate":"1"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN1_2",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN1","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN1_2 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN1_2 _NEG.raw","Replicate":"2"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN1_3",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN1","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN1_3 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN1_3 _NEG.raw","Replicate":"3"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN1_4",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN1","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN1_4 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN1_4 _NEG.raw","Replicate":"4"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN2_1",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN2","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN2_1 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN2_1 _NEG.raw","Replicate":"1"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN2_2",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN2","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN2_2 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN2_2 _NEG.raw","Replicate":"2"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN2_3",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN2","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN2_3 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN2_3 _NEG.raw","Replicate":"3"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN2_4",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN2","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN2_4 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN2_4 _NEG.raw","Replicate":"4"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN3_1",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN3","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN3_1 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN3_1 _NEG.raw","Replicate":"1"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN3_2",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN3","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN3_2 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN3_2 _NEG.raw","Replicate":"2"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN3_3",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN3","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN3_3 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN3_3 _NEG.raw","Replicate":"3"}
},
{
"Subject ID":"-",
"Sample ID":"IgAN3_4",
"Factors":{"Phenotype":"IgAN"},
"Additional sample data":{"Cell Line ID":"IgAN3","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_IgAN3_4 _POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_IgAN3_4 _NEG.raw","Replicate":"4"}
},
{
"Subject ID":"-",
"Sample ID":"Pool_1",
"Factors":{"Phenotype":"Pool"},
"Additional sample data":{"Cell Line ID":"Pool","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_Pool_1_POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_Pool_1_NEG.raw","Replicate":"1"}
},
{
"Subject ID":"-",
"Sample ID":"Pool_2",
"Factors":{"Phenotype":"Pool"},
"Additional sample data":{"Cell Line ID":"Pool","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_Pool_2_POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_Pool_2_NEG.raw","Replicate":"2"}
},
{
"Subject ID":"-",
"Sample ID":"Pool_3",
"Factors":{"Phenotype":"Pool"},
"Additional sample data":{"Cell Line ID":"Pool","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_Pool_3_POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_Pool_3_NEG.raw","Replicate":"3"}
},
{
"Subject ID":"-",
"Sample ID":"Pool_4",
"Factors":{"Phenotype":"Pool"},
"Additional sample data":{"Cell Line ID":"Pool","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_Pool_4_POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_Pool_4_NEG.raw","Replicate":"4"}
},
{
"Subject ID":"-",
"Sample ID":"Pool_5",
"Factors":{"Phenotype":"Pool"},
"Additional sample data":{"Cell Line ID":"Pool","Sample Identifer Instrument Run Name (POS)":"Nk_Test_Cells_Pool_5_POS.raw","Sample Identifier Instrument Run Name (NEG)":"Nk_Test_Cells_Pool_5_NEG.raw","Replicate":"5"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"An aliquot of cells (1 x 10^7) grown in suspension culture was transferred into a 15 mL conical tube containing 4x the culture volume of ice-cold 0.9% (w/v) NaCl. Cells were pelleted for 5 minutes at 1,500 rpm in a 4oC refrigerated centrifuge. Supernatant was removed, and the pellet was gently resuspended in 1 mL of ice-cold 0.9% (w/v) NaCl to wash and transfer into a 2 mL Lo-Bind microcentrifuge tube (Eppendorf, #022431102). Cells were pelleted for 3 minutes at 1,300 rcf in a 4oC refrigerated centrifuge. The entire supernatant was removed and pellets were snap-frozen in liquid nitrogen and immediately stored at -70°C.","SAMPLE_TYPE":"Cell pellets","STORAGE_CONDITIONS":"-80 C","TISSUE_CELL_QUANTITY_TAKEN":"1 x 10^7"},

"TREATMENT":{"TREATMENT_SUMMARY":"None","CELL_MEDIA":"10%FBS, 11mM Glucose"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"A total of 24 cell pellets (4 replicates from 6 cell lines) were shipped to the NIH RTI-RCMRC on dry ice and immediately stored at -80oC after being inventoried for metabolomics analysis. An addition of 500 µL of ice-cold Cell Extraction buffer (0.005 tryptophan-d5 in 50:50 Acetonitre:Water) was added to tubes containing the cell pellet samples on ice. MagNA Lyser ceramic beads (~15-20, prewashed & dried) were added to the tubes and the MagNA Lyser was used to beat samples for two 30 sec pulses at 2,000 rpm and samples were placed on a cold block for 1 min between pulses. Samples were then centrifuged at room temperature at 16,000 rcf for 4 min. A 15 mL washed conical tube was labeled Total Pool and 120 µL of each sample was added to this conical tube. There were 24 samples; thus, the resulting Total Pool was 2880 µL. The Total Pool was vortexed and 150 µL of the Total Pool was transferred to pre-labeled 1.5 mL Lo-Bind Eppendorf tubes to make two sets of 5 Total Pool samples, and 3 Equilibrium samples. The 150 uL of the remaining supernatants of each sample was transferred to a new, pre-labeled 1.5 mL Lo-Bind Eppendorf tubes and capped with disposable rubber stoppers. Samples were placed at -80 °C for 1 h and lyophilized overnight. 100 µL of 95:5 Acetonitrile:Water was added to each tube and vortexed for 2 min at 5000 rpm, followed by centrifugation at room temperature at 16,000 rcf for 4 min. The supernatants were transferred to pre-labeled autosampler vials and 3 µL was injected into SYNAPT G2-Si. All samples were prepared and analyzed in a randomized order."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Hydrophilic Interaction Liquid Chromatography (HILIC) Gradient Seperation","INSTRUMENT_NAME":"Waters Acquity I-Class","COLUMN_NAME":"Waters Acquity BEH Amide (150 x 2.1mm,1.7um)","CHROMATOGRAPHY_TYPE":"HILIC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Waters Synapt G2 Si QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE"}

}