Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA028921

Local Sample ID:	IgAN2_1
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

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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:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
IgAN2_1	SA028921	FL007042	IgAN	Phenotype

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 QTOF	Waters Synapt G2 Si QTOF
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 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

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