Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA162667

Local Sample ID:	POL-06
Subject ID:	SU001811
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001811
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
POL-06	SA162667	FL019221	non-allergic	group

Collection:

Collection ID:	CO001804
Collection Summary:	During endoscopic surgical procedures to remove the polyp, 5 mm biopsies of nasal polyp were obtained and kept in RNA later.
Sample Type:	Nasal Polyp tissue
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001824
Treatment Summary:	Nasal polyp samples were kept in RNA later and stored at -80ºC until preparation

Sample Preparation:

Sampleprep ID:	SP001817
Sampleprep Summary:	RNAlater solvent was removed by washing the tissue 3 times with PBS 1X. Then, the polyp was frozen in liquid nitrogen for 30 seconds. The frozen sample was put in cryoPREPTMCP02 (Covaris, MA, United States) plastic bags and submerged again for 30 seconds in liquid nitrogen. Once the plastic bag was inside the automated cryoPREPTMCP02, two consecutive impact forces of levels 2 and 4 out of 6 were applied. The resultant powder was gathered and weighted. Then, 100 µL of cold methanol:ethanol (1:1, v/v) and 0.5 µL of internal standard (LPC 18:1-d7; 0.01mM) were added per each 10 mg of tissue for metabolite extraction and protein precipitation. Samples were then vortex-mixed and homogenized using Tissue-Lyser LT homogenizer (Qiagen, Germany) for 5 min at 50 Hz, 3 times. Supernatant containing the metabolites was separated from the pellet by centrifugation (2,000 rcf for 10 min at 4 °C). Then, an aliquot of 70 µL was transferred to an LC vial and diluted with 490 µL of mobile phase (5% water: 95% acetonitrile; both with 7.5 mM ammonium acetate and 0.1% acetic acid). All samples were randomized before metabolite extraction and for the corresponding analytical run.

Sampleprep ID:

SP001817

Sampleprep Summary:

RNAlater solvent was removed by washing the tissue 3 times with PBS 1X. Then, the polyp was frozen in liquid nitrogen for 30 seconds. The frozen sample was put in cryoPREPTMCP02 (Covaris, MA, United States) plastic bags and submerged again for 30 seconds in liquid nitrogen. Once the plastic bag was inside the automated cryoPREPTMCP02, two consecutive impact forces of levels 2 and 4 out of 6 were applied. The resultant powder was gathered and weighted. Then, 100 µL of cold methanol:ethanol (1:1, v/v) and 0.5 µL of internal standard (LPC 18:1-d7; 0.01mM) were added per each 10 mg of tissue for metabolite extraction and protein precipitation. Samples were then vortex-mixed and homogenized using Tissue-Lyser LT homogenizer (Qiagen, Germany) for 5 min at 50 Hz, 3 times. Supernatant containing the metabolites was separated from the pellet by centrifugation (2,000 rcf for 10 min at 4 °C). Then, an aliquot of 70 µL was transferred to an LC vial and diluted with 490 µL of mobile phase (5% water: 95% acetonitrile; both with 7.5 mM ammonium acetate and 0.1% acetic acid). All samples were randomized before metabolite extraction and for the corresponding analytical run.

Combined analysis:

Analysis ID	AN002823
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Agilent 1260
Column	Phenomenex Kinetex HILIC 100A (50 x 2.1 mm,2.6um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Agilent 6470 QQQ
Ion Mode	NEGATIVE
Units	ug/mg

Chromatography:

Chromatography ID:	CH002087
Chromatography Summary:	We used HPLC system (1260 Infinity, Agilent Technologies). Metabolites were separated on a Kinetex hydrophilic interaction liquid chromatography (HILIC) silica column (150 mm × 2.1 mm, particle size 100Å, Phenomenex, USA) maintained at 25 ºC. The mobile phases consisted of A) water, and B) acetonitrile, both with 7.5 mM ammonium acetate and 0.1% acetic acid, with a final pH of 4.0 in the aqueous phase. The flow rate was 0.5 mL/min with an injection volume of 5 µl. Gradient started with 5% of A for 2 min, then increased up to 50% until 12 min, and back to initial conditions until 22 min.
Instrument Name:	Agilent 1260
Column Name:	Phenomenex Kinetex HILIC 100A (50 x 2.1 mm,2.6um)
Column Temperature:	25ºC
Flow Rate:	0.5 mL/min
Solvent A:	100% water; 0.1% acetic acid; 7.5 mM ammonium acetate, pH 4
Solvent B:	100% acetonitrile; 0.1% acetic acid; 7.5 mM ammonium acetate
Chromatography Type:	HILIC

MS:

MS ID:	MS002617
Analysis ID:	AN002823
Instrument Name:	Agilent 6470 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The MS conditions were: 5500 V of capillary voltage in positive ESI mode, a nebulizer gas flow rate of 11.0 L/min; a source temperature of 250 °C; and a source pressure of 60 psi. The sample tray temperature was maintained at 4 °C. Each transition was optimized adjusting the fragmentor and collision energy voltages. Data were acquired in MassHunter Workstation B.05.00 (Agilent Technologies), and re-processed using MassHunter QQQ Quantitative Analysis B.08.00 (Agilent) where peak areas were integrated. Concentration of metabolites were calculated using calibration curves with the standard addition method.
Ion Mode:	NEGATIVE