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MB Sample ID: SA181782

Local Sample ID:	N23
Subject ID:	SU002011
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	23-73
Gender:	Male and female

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Subject:

Subject ID:	SU002011
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	23-73
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
N23	SA181782	FL022281	CO	Group

Collection:

Collection ID:	CO002004
Collection Summary:	All the convalescent patients (both CA and CO groups) were discharged from the hospital after two consecutive negative results from throat swab tests for SARS-CoV-2. All patients were followed up for months after discharging and their plasma samples were collected 60 -100 days after the first onset of the disease symptoms onset. All blood samples were collected with potassium-EDTA blood collection tubes after overnight fasting. We classified the convalescent patients into the CA and CO groups according to the results of the ELISA test of anti-SARS-CoV-2 IgG following the manufacturer’s instructions.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR002023
Treatment Summary:	We classified the convalescent patients into the CA and CO groups according to the results of the ELISA test of anti-SARS-CoV-2 IgG following the manufacturer’s instructions. Briefly, 100 μL of diluted plasma samples (1:100 to 1: 800 dilution) was added to pre-coated plates, and the plates were then incubated at 37°C for 1 h. After washing, 100 μL of horseradish peroxidase (HRP)-conjugated RBD protein of SARS-CoV-2 was added into each well, followed by 30 more min of incubation at 37°C. After washing, the OD value at 450 nm (A450) was determined. The cutoff for negative was calculated by summing 0.090 and the average A450 of negative-control. A sample was determined negative when its A450 was below this cutoff value.

Treatment ID:

TR002023

Treatment Summary:

We classified the convalescent patients into the CA and CO groups according to the results of the ELISA test of anti-SARS-CoV-2 IgG following the manufacturer’s instructions. Briefly, 100 μL of diluted plasma samples (1:100 to 1: 800 dilution) was added to pre-coated plates, and the plates were then incubated at 37°C for 1 h. After washing, 100 μL of horseradish peroxidase (HRP)-conjugated RBD protein of SARS-CoV-2 was added into each well, followed by 30 more min of incubation at 37°C. After washing, the OD value at 450 nm (A450) was determined. The cutoff for negative was calculated by summing 0.090 and the average A450 of negative-control. A sample was determined negative when its A450 was below this cutoff value.

Sample Preparation:

Sampleprep ID:	SP002017
Sampleprep Summary:	Metabolomics of plasma samples were prepared and quantified as according to previously described method with modification. In brief, the standard solutions (5.0 mg/mL) were made by dissolving the accurately weighed chemicals in appropriate solutions including water, methanol, sodium hydroxide solution, or hydrochloric acid solution. Then the stock calibration solutions were mixed from the appropriate amounts of individual stock solutions following the instruction of the manufacturer. After thawing at 4°C, 20-μL aliquots of the samples were added to a 96-well plate. Also were added to the plate the calibration solutions of eight various concentrations, quality control (QC) samples (equally mixed samples), as well as solvent blank. Then 120 μL of standard solution was added to each well. The microporous plate was covered with aluminum film, placed on constant temperature mixer, and vibrated at 10℃, 650rpm for 20min. After centrifugation at 4000g for 20 min, 30μl supernatant from each well was transferred to a new 96-well plate. The derivative reagents of Q300 Kit were added to all wells and the plate was covered and incubated at 1200 rpm at 30℃ to carry out derivatization for 60 min. After derivatization, 330 μL of precooled 50% methanol solution was added to each well and mixed with the samples at 1200 rpm at 10℃ for 5 minutes. Then the plat were centrifuged at 4000g, 4°C for 30 minutes. Finally, the supernatant was further transferred to a new 96-well plate and put into the automatic sampler of UPLC-MS analysis.

Sampleprep ID:

SP002017

Sampleprep Summary:

Metabolomics of plasma samples were prepared and quantified as according to previously described method with modification. In brief, the standard solutions (5.0 mg/mL) were made by dissolving the accurately weighed chemicals in appropriate solutions including water, methanol, sodium hydroxide solution, or hydrochloric acid solution. Then the stock calibration solutions were mixed from the appropriate amounts of individual stock solutions following the instruction of the manufacturer. After thawing at 4°C, 20-μL aliquots of the samples were added to a 96-well plate. Also were added to the plate the calibration solutions of eight various concentrations, quality control (QC) samples (equally mixed samples), as well as solvent blank. Then 120 μL of standard solution was added to each well. The microporous plate was covered with aluminum film, placed on constant temperature mixer, and vibrated at 10℃, 650rpm for 20min. After centrifugation at 4000g for 20 min, 30μl supernatant from each well was transferred to a new 96-well plate. The derivative reagents of Q300 Kit were added to all wells and the plate was covered and incubated at 1200 rpm at 30℃ to carry out derivatization for 60 min. After derivatization, 330 μL of precooled 50% methanol solution was added to each well and mixed with the samples at 1200 rpm at 10℃ for 5 minutes. Then the plat were centrifuged at 4000g, 4°C for 30 minutes. Finally, the supernatant was further transferred to a new 96-well plate and put into the automatic sampler of UPLC-MS analysis.

Combined analysis:

Analysis ID	AN003143
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	The Shim-pack UFLC SHIMADZU CBM A ultrahigh-performance liquid chromatography (UHPLC) system
Column	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Ion trap
MS instrument name	ABI Sciex 6500 QTrap
Ion Mode	UNSPECIFIED
Units	µM

Chromatography:

Chromatography ID:	CH002325
Chromatography Summary:	The Shim-pack UFLC SHIMADZU CBM A ultrahigh-performance liquid chromatography (UHPLC) system (SHIMADZU, Japan) coupled with QTRAP 6500+ triple quadrupole mass spectrometer (Sciex, Washington, USA) was used to analyze the metabolomics. ACQUITY UPLC BEH C18 1.7 µm VanGuard pre-column (2.1mm × 5 mm) and ACQUITY UPLC BEH C18 1.7 µm analytical column (2.1 × 100 mm) (Waters Corporation, Milford, MA, USA) were applied to this system. The mobile phases A and B were 0.1% formic acid solution in water and acetonitrile-IPA mixture (70:30, v/v), respectively. A 5-μL injection of each sample was maintained at 40 °C and the flow rate was 0.40 mL/min. The mobile phase gradient was: 0-1 min (5% B), 1-11min (5-78% B), 11-13.5 min (78-95% B), 13.5-14 min (95-100% B), 14-16 min (100% B), 16-16.1 min (100-5% B), and 16.1-18 min (5% B).
Instrument Name:	The Shim-pack UFLC SHIMADZU CBM A ultrahigh-performance liquid chromatography (UHPLC) system
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	40
Flow Gradient:	0-1 min (5% B), 1-11min (5-78% B), 11-13.5 min (78-95% B), 13.5-14 min (95-100% B), 14-16 min (100% B), 16-16.1 min (100-5% B), and 16.1-18 min (5% B).
Flow Rate:	0.40 mL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	70% acetonitrile/30% isopropanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002923
Analysis ID:	AN003143
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Ion trap
MS Type:	ESI
MS Comments:	The Shim-pack UFLC SHIMADZU CBM A ultrahigh-performance liquid chromatography (UHPLC) system (SHIMADZU, Japan) coupled with QTRAP 6500+ triple quadrupole mass spectrometer (Sciex, Washington, USA) was used to analyze the metabolomics. ACQUITY UPLC BEH C18 1.7 µm VanGuard pre-column (2.1mm × 5 mm) and ACQUITY UPLC BEH C18 1.7 µm analytical column (2.1 × 100 mm) (Waters Corporation, Milford, MA, USA) were applied to this system. The mass spectrometer was operated in the both positive and negative modes, with capillary voltages of 1.5 and 2.0 kV, respectively. The source and desolvation temperatures were 150°C and 550 °C, respectively. The flow rate of desolvation gas was 1000 L/Hr.
Ion Mode:	UNSPECIFIED