#METABOLOMICS WORKBENCH jgooding_20170217_122401 DATATRACK_ID:847 STUDY_ID:ST000560 ANALYSIS_ID:AN000860 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Metabolomics of immunoglobulin-producing cells in IgA nephropathy PR:PROJECT_SUMMARY IgA nephropathy (IgAN), the most common primary glomerulonephritis, is PR:PROJECT_SUMMARY characterized by deposits of IgA-containing immune complexes in the kidney PR:PROJECT_SUMMARY glomeruli, as first described by Berger and Hinglais in 1968. IgAN is a major PR:PROJECT_SUMMARY cause of end-stage renal disease with its associated cardio-renal morbidity and PR:PROJECT_SUMMARY mortality. Analyses of the IgA deposits revealed that the IgA is exclusively of PR:PROJECT_SUMMARY the IgA1 subclass and that this IgA1 is aberrantly glycosylated, deficient in PR:PROJECT_SUMMARY galactose in some O-glycans (Gd-IgA1). Patients with IgAN have elevated serum PR:PROJECT_SUMMARY levels of Gd-IgA1 bound by anti-glycan autoantibodies in circulating immune PR:PROJECT_SUMMARY complexes (CIC) that are fundamental in driving disease pathology in an PR:PROJECT_SUMMARY autoimmune process. We have recently shown that elevated serum levels of Gd-IgA1 PR:PROJECT_SUMMARY in patients with IgAN predict disease progression. Thus, understanding the PR:PROJECT_SUMMARY mechanisms behind Gd-IgA1 production will improve future treatment options, as PR:PROJECT_SUMMARY there is presently no disease-specific therapy. A total of 24 cell pellets (4 PR:PROJECT_SUMMARY replicates from 6 cell lines) were analyzed by LCMS metabolomics. Immortalized PR:PROJECT_SUMMARY immunoglobulin-producing cell lines were generated from peripheral-blood PR:PROJECT_SUMMARY lymphocytes from patients with IgAN and healthy controls as described in Suzuki, PR:PROJECT_SUMMARY H., Moldoveanu, Z., Hall, S., et al. IgA1-secreting cell lines from patients PR:PROJECT_SUMMARY with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Invest. 2008, PR:PROJECT_SUMMARY 118, 629-639. PR:INSTITUTE University of Alabama, Birmingham PR:DEPARTMENT Departments of Microbiology and Medicine PR:LAST_NAME Novak PR:FIRST_NAME Jan PR:ADDRESS 845 19th St.South, BBRB 761A PR:EMAIL jannovak@uab.edu PR:PHONE 205-934-4480 PR:FUNDING_SOURCE NIH/NIGMS Grant # K01GM109320 to Jessica Gooding; NIDDK Grant # K01DK106341 to PR:FUNDING_SOURCE Colin Reily; NIDDK Grant # DK078244 to Jan Novak and NIH Common Fund ERCMRC PR:FUNDING_SOURCE Grant # U24DK097193 to Susan Sumner PR:DOI http://dx.doi.org/10.21228/M8XG7R PR:CONTRIBUTORS Jessica Gooding1,2, Colin Reily3, Courtney Whitaker1,2, Hieu Sy Vu1,2, Zach PR:CONTRIBUTORS Acuff1,2, Susan McRitchie1,2, Bruce A. Julian3, Jan Novak3, Susan Sumner2,4 PR:CONTRIBUTORS 1Analytical Chemistry & Pharmaceutics, RTI International, RTP, NC 2NIH Eastern PR:CONTRIBUTORS Regional Comprehensive Metabolomics Resource Core (ERCMRC) at UNC Chapel Hill, PR:CONTRIBUTORS NC 3Departments of Microbiology and Medicine, University of Alabama at PR:CONTRIBUTORS Birmingham, Birmingham, AL 4Nutrition Research Institute, University of North PR:CONTRIBUTORS Carolina, Chapel Hill, NC #STUDY ST:STUDY_TITLE Metabolomics of immunoglobulin-producing cells in IgA nephropathy ST:STUDY_SUMMARY IgA nephropathy (IgAN), the most common primary glomerulonephritis, is ST:STUDY_SUMMARY characterized by deposits of IgA-containing immune complexes in the kidney ST:STUDY_SUMMARY glomeruli, as first described by Berger and Hinglais in 1968. IgAN is a major ST:STUDY_SUMMARY cause of end-stage renal disease with its associated cardio-renal morbidity and ST:STUDY_SUMMARY mortality. Analyses of the IgA deposits revealed that the IgA is exclusively of ST:STUDY_SUMMARY the IgA1 subclass and that this IgA1 is aberrantly glycosylated, deficient in ST:STUDY_SUMMARY galactose in some O-glycans (Gd-IgA1). Patients with IgAN have elevated serum ST:STUDY_SUMMARY levels of Gd-IgA1 bound by anti-glycan autoantibodies in circulating immune ST:STUDY_SUMMARY complexes (CIC) that are fundamental in driving disease pathology in an ST:STUDY_SUMMARY autoimmune process. We have recently shown that elevated serum levels of Gd-IgA1 ST:STUDY_SUMMARY in patients with IgAN predict disease progression. Thus, understanding the ST:STUDY_SUMMARY mechanisms behind Gd-IgA1 production will improve future treatment options, as ST:STUDY_SUMMARY there is presently no disease-specific therapy. A total of 24 cell pellets (4 ST:STUDY_SUMMARY replicates from 6 cell lines) were analyzed by LCMS metabolomics. Immortalized ST:STUDY_SUMMARY immunoglobulin-producing cell lines were generated from peripheral-blood ST:STUDY_SUMMARY lymphocytes from patients with IgAN and healthy controls as described in Suzuki, ST:STUDY_SUMMARY H., Moldoveanu, Z., Hall, S., et al. IgA1-secreting cell lines from patients ST:STUDY_SUMMARY with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Invest. 2008, ST:STUDY_SUMMARY 118, 629-639. ST:INSTITUTE RTI International ST:LABORATORY NIH Eastern Regional Comphrehensive Metabolomics Resource Core at UNC Chapel ST:LABORATORY Hill (ERCMRC) ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 3040 E. Cornwallis Road, Research Triangle Park, NC 27709 ST:EMAIL susan_sumner@unc.edu ST:PHONE 704-250-5000 ST:SUBMIT_DATE 2017-02-17 #SUBJECT SU:SUBJECT_TYPE Cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS Suzuki, H., Moldoveanu, Z., Hall, S., et al. IgA1-secreting cell lines from SU:CELL_STRAIN_DETAILS patients with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Inv. SU:CELL_STRAIN_DETAILS 2008, 118, 629-639 SU:CELL_PRIMARY_IMMORTALIZED immortalized SU:CELL_COUNTS 1x10^7 cells / pellet SU:SPECIES_GROUP Human #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - HC1_1 Phenotype:HC 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_SAMPLE_FACTORS - HC1_2 Phenotype:HC 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_SAMPLE_FACTORS - HC1_3 Phenotype:HC 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_SAMPLE_FACTORS - HC1_4 Phenotype:HC 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_SAMPLE_FACTORS - HC2_1 Phenotype:HC 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_SAMPLE_FACTORS - HC2_2 Phenotype:HC 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_SAMPLE_FACTORS - HC2_3 Phenotype:HC 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_SAMPLE_FACTORS - HC2_4 Phenotype:HC 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_SAMPLE_FACTORS - HC3_1 Phenotype:HC 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_SAMPLE_FACTORS - HC3_2 Phenotype:HC 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_SAMPLE_FACTORS - HC3_3 Phenotype:HC 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_SAMPLE_FACTORS - HC3_4 Phenotype:HC 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_SAMPLE_FACTORS - IgAN1_1 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN1_2 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN1_3 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN1_4 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN2_1 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN2_2 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN2_3 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN2_4 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN3_1 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN3_2 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN3_3 Phenotype:IgAN 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_SAMPLE_FACTORS - IgAN3_4 Phenotype:IgAN 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_SAMPLE_FACTORS - Pool_1 Phenotype:Pool 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_SAMPLE_FACTORS - Pool_2 Phenotype:Pool 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_SAMPLE_FACTORS - Pool_3 Phenotype:Pool 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_SAMPLE_FACTORS - Pool_4 Phenotype:Pool 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_SAMPLE_FACTORS - Pool_5 Phenotype:Pool 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 CO:COLLECTION_SUMMARY An aliquot of cells (1 x 10^7) grown in suspension culture was transferred into CO:COLLECTION_SUMMARY a 15 mL conical tube containing 4x the culture volume of ice-cold 0.9% (w/v) CO:COLLECTION_SUMMARY NaCl. Cells were pelleted for 5 minutes at 1,500 rpm in a 4oC refrigerated CO:COLLECTION_SUMMARY centrifuge. Supernatant was removed, and the pellet was gently resuspended in 1 CO:COLLECTION_SUMMARY mL of ice-cold 0.9% (w/v) NaCl to wash and transfer into a 2 mL Lo-Bind CO:COLLECTION_SUMMARY microcentrifuge tube (Eppendorf, #022431102). Cells were pelleted for 3 minutes CO:COLLECTION_SUMMARY at 1,300 rcf in a 4oC refrigerated centrifuge. The entire supernatant was CO:COLLECTION_SUMMARY removed and pellets were snap-frozen in liquid nitrogen and immediately stored CO:COLLECTION_SUMMARY at -70°C. CO:SAMPLE_TYPE Cell pellets CO:STORAGE_CONDITIONS -80 C CO:TISSUE_CELL_QUANTITY_TAKEN 1 x 10^7 #TREATMENT TR:TREATMENT_SUMMARY None TR:CELL_MEDIA 10%FBS, 11mM Glucose #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A total of 24 cell pellets (4 replicates from 6 cell lines) were shipped to the SP:SAMPLEPREP_SUMMARY NIH RTI-RCMRC on dry ice and immediately stored at -80oC after being inventoried SP:SAMPLEPREP_SUMMARY for metabolomics analysis. An addition of 500 µL of ice-cold Cell Extraction SP:SAMPLEPREP_SUMMARY buffer (0.005 tryptophan-d5 in 50:50 Acetonitre:Water) was added to tubes SP:SAMPLEPREP_SUMMARY containing the cell pellet samples on ice. MagNA Lyser ceramic beads (~15-20, SP:SAMPLEPREP_SUMMARY prewashed & dried) were added to the tubes and the MagNA Lyser was used to beat SP:SAMPLEPREP_SUMMARY samples for two 30 sec pulses at 2,000 rpm and samples were placed on a cold SP:SAMPLEPREP_SUMMARY block for 1 min between pulses. Samples were then centrifuged at room SP:SAMPLEPREP_SUMMARY temperature at 16,000 rcf for 4 min. A 15 mL washed conical tube was labeled SP:SAMPLEPREP_SUMMARY Total Pool and 120 µL of each sample was added to this conical tube. There were SP:SAMPLEPREP_SUMMARY 24 samples; thus, the resulting Total Pool was 2880 µL. The Total Pool was SP:SAMPLEPREP_SUMMARY vortexed and 150 µL of the Total Pool was transferred to pre-labeled 1.5 mL SP:SAMPLEPREP_SUMMARY Lo-Bind Eppendorf tubes to make two sets of 5 Total Pool samples, and 3 SP:SAMPLEPREP_SUMMARY Equilibrium samples. The 150 uL of the remaining supernatants of each sample was SP:SAMPLEPREP_SUMMARY transferred to a new, pre-labeled 1.5 mL Lo-Bind Eppendorf tubes and capped with SP:SAMPLEPREP_SUMMARY disposable rubber stoppers. Samples were placed at -80 °C for 1 h and SP:SAMPLEPREP_SUMMARY lyophilized overnight. 100 µL of 95:5 Acetonitrile:Water was added to each tube SP:SAMPLEPREP_SUMMARY and vortexed for 2 min at 5000 rpm, followed by centrifugation at room SP:SAMPLEPREP_SUMMARY temperature at 16,000 rcf for 4 min. The supernatants were transferred to SP:SAMPLEPREP_SUMMARY pre-labeled autosampler vials and 3 µL was injected into SYNAPT G2-Si. All SP:SAMPLEPREP_SUMMARY samples were prepared and analyzed in a randomized order. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Hydrophilic Interaction Liquid Chromatography (HILIC) Gradient Seperation CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity BEH Amide (150 x 2.1mm,1.7um) CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE #END