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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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.