Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA257915

Local Sample ID:	SR01
Subject ID:	SU002665
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	WT/PMM2-CDG
Age Or Age Range:	5-45
Gender:	Male and female

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Subject:

Subject ID:	SU002665
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	WT/PMM2-CDG
Age Or Age Range:	5-45
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
SR01	SA257915	FL033113	PMM2-CDG	Genotype
SR01	SA257915	FL033113	5.5mM 13C GLU negative siRNA	Treatment

Collection:

Collection ID:	CO002658
Collection Summary:	Briefly, medium was removed from the cells and the cells were washed 3 times with 1 mL Dulbecco PBS containing 0.901 mM CaCl2 (Merck) and 0.492 mM MgCl2 (Merck). Next, cells were incubated with 1 mg/100 mL EZ-Link-Sulfo-NHS-LC-Biotin (Thermo) in Dulbecco for 30 min, RT, shaking. Cells were then washed twice with 1 mL Dulbecco, non-reacted biotin was blocked with 1 mL 20 mM glycine in Dulbecco for 15 min, and cells washed again with 1 mL Dulbecco. Dulbecco was then removed, cells scraped in 200 µL RIPA buffer (with protease inhibitors) and transferred to a fresh Eppendorf tube. Samples were lysed on ice with 3 consecutive freeze-thaw cycles. Further, 30 µL dynabeads streptavidin T1 (Invitrogen) was added to each sample and the samples were washed twice with 500 µL 10 mM ammonium bicarbonate and neuraminidase buffer (100 mM Sodium Acetate Buffer with 2 mM CaCl2 (Merck), pH 5.0). Neuraminidase buffer was removed and 500 µL PBS was added to the beads. 30 µL of prepared mix was added to each sample, and samples were incubated, shaking overnight at 4 °C. Samples were put on a dynabead rack (Invitrogen) and the supernatant was transferred to a new Eppendorf tube and used for protein concentration assay. Beads containing membrane fractions were washed 2 times with 500 µL lysis buffer (2 % IGEPAL (Sigma), 1% Triton X-100 (Sigma), and 10 % glycerol inPBS) and then washed with 1 mL PBS. Samples are centrifuged at 500 rcf, 5 min, 4 °C and PBS was removed. 100 µL neuraminidase buffer containing 0.05 U neuraminidase was added to each sample and samples were incubated overnight at 37 °C, shaking. Next, supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. Finally, pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, IS). Sialic acid was measured by LC/MS as described below. El Maven Polly software was used to annotate sialic acid based on m/z ratio and elution time, and determine fractional contribution of glucose in sialic acid.
Sample Type:	Fibroblasts
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002677
Treatment Summary:	Cells were treated with vehicle or 5nM siRNA targeting AKR1B1, Before collection, medium was removed from the cells and the cells were washed 3 times with 1 mL Dulbecco PBS containing 0.901 mM CaCl2 (Merck) and 0.492 mM MgCl2 (Merck). Next, cells were incubated with 1 mg/100 mL EZ-Link-Sulfo-NHS-LC-Biotin (Thermo) in Dulbecco for 30 min, RT, shaking. Cells were then washed twice with 1 mL Dulbecco, non-reacted biotin was blocked with 1 mL 20 mM glycine in Dulbecco for 15 min, and cells washed again with 1 mL Dulbecco. Dulbecco was then removed, cells scraped in 200 µL RIPA buffer (with protease inhibitors) and transferred to a fresh Eppendorf tube. Samples were lysed on ice with 3 consecutive freeze-thaw cycles. Further, 30 µL dynabeads streptavidin T1 (Invitrogen) was added to each sample and the samples were washed twice with 500 µL 10 mM ammonium bicarbonate and neuraminidase buffer (100 mM Sodium Acetate Buffer with 2 mM CaCl2 (Merck), pH 5.0). Neuraminidase buffer was removed and 500 µL PBS was added to the beads. 30 µL of prepared mix was added to each sample, and samples were incubated, shaking overnight at 4 °C. Samples were put on a dynabead rack (Invitrogen) and the supernatant was transferred to a new Eppendorf tube and used for protein concentration assay. Beads containing membrane fractions were washed 2 times with 500 µL lysis buffer (2 % IGEPAL (Sigma), 1% Triton X-100 (Sigma), and 10 % glycerol inPBS) and then washed with 1 mL PBS. Samples are centrifuged at 500 rcf, 5 min, 4 °C and PBS was removed. 100 µL neuraminidase buffer containing 0.05 U neuraminidase was added to each sample and samples were incubated overnight at 37 °C, shaking. Next, supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. Finally, pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, IS). Sialic acid was measured by LC/MS as described below. El Maven Polly software was used to annotate sialic acid based on m/z ratio and elution time, and determine fractional contribution of glucose in sialic acid.

Treatment ID:

TR002677

Treatment Summary:

Cells were treated with vehicle or 5nM siRNA targeting AKR1B1, Before collection, medium was removed from the cells and the cells were washed 3 times with 1 mL Dulbecco PBS containing 0.901 mM CaCl2 (Merck) and 0.492 mM MgCl2 (Merck). Next, cells were incubated with 1 mg/100 mL EZ-Link-Sulfo-NHS-LC-Biotin (Thermo) in Dulbecco for 30 min, RT, shaking. Cells were then washed twice with 1 mL Dulbecco, non-reacted biotin was blocked with 1 mL 20 mM glycine in Dulbecco for 15 min, and cells washed again with 1 mL Dulbecco. Dulbecco was then removed, cells scraped in 200 µL RIPA buffer (with protease inhibitors) and transferred to a fresh Eppendorf tube. Samples were lysed on ice with 3 consecutive freeze-thaw cycles. Further, 30 µL dynabeads streptavidin T1 (Invitrogen) was added to each sample and the samples were washed twice with 500 µL 10 mM ammonium bicarbonate and neuraminidase buffer (100 mM Sodium Acetate Buffer with 2 mM CaCl2 (Merck), pH 5.0). Neuraminidase buffer was removed and 500 µL PBS was added to the beads. 30 µL of prepared mix was added to each sample, and samples were incubated, shaking overnight at 4 °C. Samples were put on a dynabead rack (Invitrogen) and the supernatant was transferred to a new Eppendorf tube and used for protein concentration assay. Beads containing membrane fractions were washed 2 times with 500 µL lysis buffer (2 % IGEPAL (Sigma), 1% Triton X-100 (Sigma), and 10 % glycerol inPBS) and then washed with 1 mL PBS. Samples are centrifuged at 500 rcf, 5 min, 4 °C and PBS was removed. 100 µL neuraminidase buffer containing 0.05 U neuraminidase was added to each sample and samples were incubated overnight at 37 °C, shaking. Next, supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. Finally, pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, IS). Sialic acid was measured by LC/MS as described below. El Maven Polly software was used to annotate sialic acid based on m/z ratio and elution time, and determine fractional contribution of glucose in sialic acid.

Sample Preparation:

Sampleprep ID:	SP002671
Sampleprep Summary:	After treatment with neuraminidase, supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. Finally, lyophilized pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, IS).
Processing Storage Conditions:	-80℃
Extract Storage:	-80℃

Combined analysis:

Analysis ID	AN004225
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	NEGATIVE
Units	AUC

Chromatography:

Chromatography ID:	CH003134
Chromatography Summary:	C18 iP REVERSE PHASE
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um)
Column Temperature:	40
Flow Gradient:	The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min post injection. A linear gradient to 37% B was carried out until 7 min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min.
Flow Rate:	0.25 mL/min
Solvent A:	100% water; 10mM tributylamine; 15mM acetic acid
Solvent B:	100% methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003972
Analysis ID:	AN004225
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	El-Maven polly, ThermoFisher Xcalibur; Sialic acid was annotated using the inhouse standard metabolite library- elution time and m/z values.
Ion Mode:	NEGATIVE