Summary of Study ST000980

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 PR000670. The data can be accessed directly via it's Project DOI: 10.21228/M82Q3W This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST000980
Study Title	Metabolomic Analysis of plasma samples from Non-Allergic Subjects and Profilin-Allergic Patients overexposed to Grass Pollen
Study Type	Allergy severity comparison
Study Summary	Prevalence and severity of allergic diseases have increased worldwide. To date, respiratory allergy phenotypes are not fully characterized and, along with inflammation progression, treatment is increasingly complex and expensive. Profilin sensitization constitutes a good model to study the progression of allergic inflammation. Our aim was to identify the underlying mechanisms and the associated biomarkers of this progression, focusing on severe phenotypes, using transcriptomics and metabolomics. Methods: 25 subjects were included in the study. Plasma samples were analyzed using Gas and Liquid Chromatography coupled to Mass Spectrometry (GC-MS and LC-MS, respectively). Individuals were classified in 4 groups – “non-allergic”, “mild”, “moderate” and “severe” – based on their clinical history, their response to an oral challenge test with profilin, and after a refinement using a mathematical metabolomic model. PBMCs were used for microarray analysis.
Institute	The Centre of Metabolomics and Bioanalysis
Department	Analytical chemistry
Last Name	Obeso Montero
First Name	David
Address	Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España
Email	david.obesomontero@beca.ceu.es
Phone	913724769
Submit Date	2018-06-13
Num Groups	4 groups
Total Subjects	25 plasma samples
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	GC-MS/LC-MS
Release Date	2018-07-17
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR000670
Project DOI:	doi: 10.21228/M82Q3W
Project Title:	Metabolomics analysis of plasma samples from non-allergic subjects and patients with different severity of food allergy
Project Summary:	Metabolomic analysis of plasma samples from grass pollen allergic patients with different levels of allergic severity to profilin.
Institute:	Institute of Applied Molecular Medicine;The Centre of Metabolomics and Bioanalysis
Department:	Analytical chemistry
Last Name:	Barber Hernández
First Name:	Domingo
Address:	Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España
Email:	domingo.barberhernandez@ceu.es
Phone:	+34 91 372 47 00 ext.4662

Subject:

Subject ID:	SU001019
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Classification
SA059957	01_002	Mild
SA059958	02_002	Mild
SA059959	04_001	Mild
SA059960	01_001	Mild
SA059961	02_003	Mild
SA059962	02_004	Moderate
SA059963	04_002	Moderate
SA059964	03_098	Moderate
SA059965	03_004	Moderate
SA059966	01_005	Moderate
SA059967	02_001	Moderate
SA059968	02_006	Non-allergic
SA059969	02_005	Non-allergic
SA059970	02_008	Non-allergic
SA059971	02_007	Non-allergic
SA059972	04_003	Non-allergic
SA059973	04_006	Non-allergic
SA059974	03_096	Severe
SA059975	03_099	Severe
SA059976	03_095	Severe
SA059977	03_003	Severe
SA059978	03_002	Severe
SA059979	03_092	Severe
SA059980	03_093	Severe
SA059981	03_094	Severe

Showing results 1 to 25 of 25

Showing results 1 to 25 of 25

Collection:

Collection ID:	CO001013
Collection Summary:	Whole blood was drawn before the profilin challenge and was divided into two tubes: one for plasma extraction and other for PBMCs isolation. Briefly, 20 ml of peripheral blood were collected to obtain plasma by centrifugation(1500rpm x 5 min.) and PBMCs using Ficoll-Paque (GE Healthcare™) density gradient centrifugation.
Sample Type:	Blood (whole)
Collection Location:	Hospital Universitario Clínico San Carlos, Madrid, España. Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IP), Madrid, España. Hospital Virgen del Puerto, Plasencia, Cáceres, España. Hospital Universitario HM Sanchinarro, Madrid, España.
Volumeoramount Collected:	14-16 mL Blood
Storage Conditions:	-80℃
Collection Vials:	BD Vacutainer® Heparin Blood Collection Tubes (Ref. BD367871)
Storage Vials:	Eppendorf 1.5 mL
Collection Tube Temp:	Room Temperature

Treatment:

Treatment ID:	TR001033
Treatment Summary:	No treatment was used in this study.

Sample Preparation:

Sampleprep ID:	SP001026
Sampleprep Summary:	For LC-MS, plasma protein was removed adding 300 µL of cold (-20 °C) methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and stored on ice for 5 min. Supernatant containing the metabolites was separated from the pellet by centrifugation (16,000× g for 20 min at 4 °C), and put into a LC vial for analysis. For GC-MS protein precipitation, 120 μL of cold acetonitrile (ACN) were added to 40 μL of each plasma sample in an Eppendorf and were stored on ice for 5 minutes. Then, metabolites were separated by centrifugation (16,000× g, 15 min, 4 °C). From the resulting supernatant, 100 μL were transferred to GC vial with insert and were evaporated to dryness (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Then, 10 μL of O-methoxyamine hydrochloride in pyridine (15 mg/mL) was added to each GC vial, and the mixture was vigorously vortex-mixed and ultrasonicated. Methoxymation was carried out in darkness, at room temperature for 16 h. Afterwards, 10 μL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) were added as catalyst and the solution was further mixed using the vortex. For silylation process, samples were heated in an oven for 1h at 70 °C. Finally, 100 μL of heptane containing 10 ppm of C18:0 methyl ester (IS) were added to each GC vial and vortex-mixed before GC analysis.

Sampleprep ID:

SP001026

Sampleprep Summary:

For LC-MS, plasma protein was removed adding 300 µL of cold (-20 °C) methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and stored on ice for 5 min. Supernatant containing the metabolites was separated from the pellet by centrifugation (16,000× g for 20 min at 4 °C), and put into a LC vial for analysis. For GC-MS protein precipitation, 120 μL of cold acetonitrile (ACN) were added to 40 μL of each plasma sample in an Eppendorf and were stored on ice for 5 minutes. Then, metabolites were separated by centrifugation (16,000× g, 15 min, 4 °C). From the resulting supernatant, 100 μL were transferred to GC vial with insert and were evaporated to dryness (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Then, 10 μL of O-methoxyamine hydrochloride in pyridine (15 mg/mL) was added to each GC vial, and the mixture was vigorously vortex-mixed and ultrasonicated. Methoxymation was carried out in darkness, at room temperature for 16 h. Afterwards, 10 μL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) were added as catalyst and the solution was further mixed using the vortex. For silylation process, samples were heated in an oven for 1h at 70 °C. Finally, 100 μL of heptane containing 10 ppm of C18:0 methyl ester (IS) were added to each GC vial and vortex-mixed before GC analysis.

Combined analysis:

Analysis ID	AN001604	AN001605	AN001606
Analysis type	MS	MS	MS
Chromatography type	Normal phase	Normal phase	GC
Chromatography system	Agilent 1200	Agilent 1200	Agilent 7890A
Column	Unspecified	Unspecified	Unspecified
MS Type	ESI	ESI	EI
MS instrument type	QTOF	QTOF	Single quadrupole
MS instrument name	Agilent 6520 QTOF	Agilent 6520 QTOF	Agilent 5975C
Ion Mode	POSITIVE	NEGATIVE	POSITIVE
Units	peak area	peak area	Peak area

Chromatography:

Chromatography ID:	CH001126
Chromatography Summary:	For LC-MS, 10 μL of sample were injected into a Discovery HS C18 column (2.1 mm × 150 mm, 3.0 μm; Supelco, Sigma Aldrich, Germany), with a guard column Discovery® HS C18 (2 cm × 2.1 mm, 3 μm; Supelco, Sigma Aldrich, Germany), both maintained at 40 °C. The flow rate was set at 0.6 mL/min. The gradient elution involved a mobile phase consisting of (A) 0.1% formic acid (FA) in water and (B) 0.1% FA in acetonitrile (ACN). The initial condition was set at 25% B, increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally it was held for 9 min in initial conditions. Flow rate was set at 0.6 mL/min, and 10 μL of samples were injected. The electrospray source ionization (ESI) data were acquired in positive and negative ion mode, respectively. The capillary voltage was set at 3,500 for ESI (+) and 4,000V for ESI (−). The drying gas flow rate was 10.5 L/min at 330 °C and gas nebulizer at 52 psi; fragmentor voltage was 175 V; skimmer and octopole radio frequency voltage (OCT RF Vpp) were set to 65 and 750 V. Data were collected in the centroid mode at a scan rate of 1.2 spectra per second. Mass spectrometry detection was performed in full scan from 100 to 1200 m/z for both, positive and negative ESI mode. The reference m/z ions were purine (121.0508) and HP-0921 (922.0097) for ESI (+), whereas TFA NH4 (119.0363) and HP-0921 (966.0007) for ESI (−). These masses were continuously infused into the system to allow constant mass correction. Samples were analyzed in separate runs.
Instrument Name:	Agilent 1200
Column Name:	Unspecified
Chromatography Type:	Normal phase

Chromatography ID:	CH001127
Chromatography Summary:	For LC-MS, 10 μL of sample were injected into a Discovery HS C18 column (2.1 mm × 150 mm, 3.0 μm; Supelco, Sigma Aldrich, Germany), with a guard column Discovery® HS C18 (2 cm × 2.1 mm, 3 μm; Supelco, Sigma Aldrich, Germany), both maintained at 40 °C. The flow rate was set at 0.6 mL/min. The gradient elution involved a mobile phase consisting of (A) 0.1% formic acid (FA) in water and (B) 0.1% FA in acetonitrile (ACN). The initial condition was set at 25% B, increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally it was held for 9 min in initial conditions. Flow rate was set at 0.6 mL/min, and 10 μL of samples were injected. The electrospray source ionization (ESI) data were acquired in positive and negative ion mode, respectively. The capillary voltage was set at 3,500 for ESI (+) and 4,000V for ESI (−). The drying gas flow rate was 10.5 L/min at 330 °C and gas nebulizer at 52 psi; fragmentor voltage was 175 V; skimmer and octopole radio frequency voltage (OCT RF Vpp) were set to 65 and 750 V. Data were collected in the centroid mode at a scan rate of 1.2 spectra per second. Mass spectrometry detection was performed in full scan from 100 to 1200 m/z for both, positive and negative ESI mode. The reference m/z ions were purine (121.0508) and HP-0921 (922.0097) for ESI (+), whereas TFA NH4 (119.0363) and HP-0921 (966.0007) for ESI (−). These masses were continuously infused into the system to allow constant mass correction. Samples were analyzed in separate runs.
Instrument Name:	Agilent 1200
Column Name:	Unspecified
Chromatography Type:	Normal phase

Chromatography ID:	CH001128
Chromatography Summary:	GC-MS analysis was performed by a GC system (Agilent Technologies 7890A) equipped with an autosampler (Agilent 7693) coupled to a mass spectrometer with triple-Axis detector (5975C, Agilent). Two microliters (2 μL) of the derivatized sample were injected through a GC-Column DB5-MS (30 m length, 0.25 mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent). Carrier gas (He) flow rate was set at 1 mL/min and injector temperature at 250 °C. Split ratio was fixed from 1:5 to 1:10 with 3 to 10 mL/min. He split flow into a Restek 20782 (Bellefonte, PA, USA) deactivated glass-wool split liner. The temperature gradient was programmed as follows: the initial oven temperature was set at 60 ºC (held for 1 min), increased to 325 ºC at 10 ºC/min rate (within 26.5 min) and hold 325 ºC for 10 min. The total run time was 37.5 min. A cool-down period was applied for 10 min before the next injection. Detector transfer line, filament source and the quadrupole temperature were set at 280 ºC, 230 ºC and 150 ºC, respectively. MS detection was performed with electron ionization (EI) mode at -70 eV. The mass spectrometer was operated in scan mode over a mass range of m/z 50-600 at a rate of 2.7 scan/s. Several IS injections, a standard mix of fatty acid methyl esters (FAME C8-C30), extraction blanks and 3 QCs samples were injected at the beginning of analysis, following QCs injections every 5 experimental samples and 1 QC injection at the end of worklist.
Instrument Name:	Agilent 7890A
Column Name:	Unspecified
Chromatography Type:	GC

MS:

MS ID:	MS001482
Analysis ID:	AN001604
Instrument Name:	Agilent 6520 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE
Capillary Voltage:	3500 V
Dry Gas Flow:	10.5 L/min
Dry Gas Temp:	330 °C
Fragment Voltage:	175 V

MS ID:	MS001483
Analysis ID:	AN001605
Instrument Name:	Agilent 6520 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE
Capillary Voltage:	4000 V
Dry Gas Flow:	10.5 L/min
Dry Gas Temp:	331 °C
Fragment Voltage:	176 V

MS ID:	MS001484
Analysis ID:	AN001606
Instrument Name:	Agilent 5975C
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE
Helium Flow:	1 mL/min
Ionization Energy:	-70 eV