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MB Sample ID: SA059968
| Local Sample ID: | 02_006 |
| Subject ID: | SU001019 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001019 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 02_006 | SA059968 | FL010547 | Non-allergic | Classification |
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. |
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 |
