Summary of Study ST000560

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 PR000411. The data can be accessed directly via it's Project DOI: 10.21228/M8XG7R This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST000560
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
Num Groups	2
Total Subjects	24
Raw Data Available	Yes
Raw Data File Type(s)	raw(Waters)
Analysis Type Detail	LC-MS
Release Date	2018-04-10
Release Version	1

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Project:

Project ID:	PR000411
Project DOI:	doi: 10.21228/M8XG7R
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
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

Subject:

Subject ID:	SU000582
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

Factors:

Subject type: Cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Phenotype
SA028898	HC3_2	HC
SA028899	HC3_3	HC
SA028900	HC3_4	HC
SA028901	HC2_4	HC
SA028902	HC1_1	HC
SA028903	HC3_1	HC
SA028904	HC2_3	HC
SA028905	HC1_2	HC
SA028906	HC1_4	HC
SA028907	HC1_3	HC
SA028908	HC2_1	HC
SA028909	HC2_2	HC
SA028910	IgAN3_2	IgAN
SA028911	IgAN3_1	IgAN
SA028912	IgAN2_4	IgAN
SA028913	IgAN3_4	IgAN
SA028914	IgAN3_3	IgAN
SA028915	IgAN1_3	IgAN
SA028916	IgAN2_3	IgAN
SA028917	IgAN1_1	IgAN
SA028918	IgAN1_2	IgAN
SA028919	IgAN1_4	IgAN
SA028920	IgAN2_2	IgAN
SA028921	IgAN2_1	IgAN
SA028922	Pool_5	Pool
SA028923	Pool_4	Pool
SA028924	Pool_1	Pool
SA028925	Pool_2	Pool
SA028926	Pool_3	Pool

Showing results 1 to 29 of 29

Showing results 1 to 29 of 29

Collection:

Collection ID:	CO000576
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 ID:	TR000596
Treatment Summary:	None
Cell Media:	10%FBS, 11mM Glucose

Sample Preparation:

Sampleprep ID:	SP000589
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.

Sampleprep ID:

SP000589

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.

Combined analysis:

Analysis ID	AN000860	AN000861
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Waters Acquity I-Class	Waters Acquity I-Class
Column	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Waters Synapt-G2-Si	Waters Synapt-G2-Si
Ion Mode	POSITIVE	NEGATIVE
Units	normalized ion counts	normalized ion counts

Chromatography:

Chromatography ID:	CH000612
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

MS:

MS ID:	MS000761
Analysis ID:	AN000860
Instrument Name:	Waters Synapt-G2-Si
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS000762
Analysis ID:	AN000861
Instrument Name:	Waters Synapt-G2-Si
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE