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MB Sample ID: SA099449
| Local Sample ID: | P3_t0 |
| Subject ID: | SU001443 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001443 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| P3_t0 | SA099449 | FL014070 | Placebo | Treatment |
| P3_t0 | SA099449 | FL014070 | Poli | Sensitization |
Collection:
| Collection ID: | CO001438 |
| Collection Summary: | For immunological analyses and metabolomics, serum samples were obtained and stored at -20°C until analysis. In the case of transcriptomics, Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and stored at -20°C in Buffer RLT until analysis. |
| Sample Type: | Blood (serum) |
| Storage Conditions: | -20℃ |
Treatment:
| Treatment ID: | TR001458 |
| Treatment Summary: | Subjects were randomized (1:1) during autumn 2013 to receive either active treatment with GRAZAX® (Phleum pratense, 75,000SQ‐T tablets ALK, Hørsholm, Denmark) or placebo as Investigational Medical Products (IMP) during 2 years. Twenty-five patients were assigned to daily sublingual administration of GRAZAX®, while the rest received placebo tablets that were similar to the active IMP with regard to appearance, smell and taste. |
Sample Preparation:
| Sampleprep ID: | SP001451 |
| Sampleprep Summary: | For LC-MS, proteins were 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, serum samples were first deproteinized using cold acetonitrile (ACN) in a 3:1 proportion (120 μL of ACN were added to 40 μL of serum). Samples were kept on ice for 5 minutes. Afterwards, the metabolites were separated by centrifugation (10 min at 15,400 x g and 4°C). 100 µL of the resulting supernatant were transferred to a GC vial with insert and were evaporated to dryness for 2 hours at 30°C (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, a methoximation was performed by addition of 10 µL of O-methoxyamine hydrochloride 15mg/mL in pyridine, with the purpose of protecting the reactive oxygen groups in the metabolites. The mixture was vigorously vortex-mixed (1 minute each vial), ultrasonicated (3 times, 20 seconds each) and further vortexed another 2 minutes. Then, the samples were left in darkness at room temperature for 16 h for the completion of the methoximation reaction. Finally, the samples were derivatized by the addition of 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS). |
| Processing Method: | Protein precipitation and metabolite extraction |
| Processing Storage Conditions: | On ice |
Combined analysis:
| Analysis ID | AN002283 | AN002284 | AN002285 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | GC |
| Chromatography system | Agilent 1200 | Agilent 1200 | Agilent 7890A |
| Column | Discovery HS C18 (150 x 2.1mm,3.0um) Supelco,Sigma Aldrich,Germany),with a guard Discovery HS C18 (2 cm x 2.1mm,3um; Supelco,Sigma Aldrich,Germany), | Discovery HS C18 (150 x 2.1mm,3.0um) Supelco,Sigma Aldrich,Germany) | 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) |
| 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: | CH001677 |
| 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: | Discovery HS C18 (150 x 2.1mm,3.0um) Supelco,Sigma Aldrich,Germany),with a guard Discovery HS C18 (2 cm x 2.1mm,3um; Supelco,Sigma Aldrich,Germany), |
| Column Temperature: | 40 |
| Flow Gradient: | 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: | 0.6ml/min |
| Injection Temperature: | 4 |
| Sample Injection: | 10 μL |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Analytical Time: | 45 min |
| Capillary Voltage: | 3500 for ESI (+) |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001678 |
| 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: | Discovery HS C18 (150 x 2.1mm,3.0um) Supelco,Sigma Aldrich,Germany) |
| Column Temperature: | 40 |
| Flow Gradient: | 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: | 0.6ml/min |
| Injection Temperature: | 4 |
| Sample Injection: | 10 μL |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Analytical Time: | 45 min |
| Capillary Voltage: | 4000V for ESI (−) |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001679 |
| 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: | 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) |
| Column Temperature: | As Oven |
| Injection Temperature: | 250 |
| Internal Standard: | Standard mix of fatty acid methyl esters (FAME C8-C30) |
| Sample Injection: | 2 μL |
| Analytical Time: | 37.5 min |
| Oven Temperature: | 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. |
| Transferline Temperature: | 280 |
| Chromatography Type: | GC |
MS:
| MS ID: | MS002127 |
| Analysis ID: | AN002283 |
| Instrument Name: | Agilent 6520 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | LC−MS analysis was performed on an Agilent HPLC system (1200 series, Agilent Technologies, Waldbronn, Germany), equipped with a degasser, two binary pumps, and a thermostated autosampler coupled with quadrupole-time of flight analyzer (Q-TOF), LC-MS (6520) system (Agilent Technologies, Waldbronn, Germany) |
| Ion Mode: | POSITIVE |
| Capillary Voltage: | 3,500 for ESI (+) |
| Dry Gas Flow: | 10.5 L/min |
| Dry Gas Temp: | 330 °C |
| Fragment Voltage: | 175 V |
| Nebulizer: | 52 psi |
| Octpole Voltage: | 750V |
| MS ID: | MS002128 |
| Analysis ID: | AN002284 |
| Instrument Name: | Agilent 6520 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | LC−MS analysis was performed on an Agilent HPLC system (1200 series, Agilent Technologies, Waldbronn, Germany), equipped with a degasser, two binary pumps, and a thermostated autosampler coupled with quadrupole-time of flight analyzer (Q-TOF), LC-MS (6520) system (Agilent Technologies, Waldbronn, Germany) |
| Ion Mode: | NEGATIVE |
| Capillary Voltage: | 4,000V for ESI (−) |
| Dry Gas Flow: | 10.5 L/min |
| Dry Gas Temp: | 330 °C |
| Fragment Voltage: | 175 V |
| Nebulizer: | 52 psi |
| Octpole Voltage: | 750V |
| MS ID: | MS002129 |
| Analysis ID: | AN002285 |
| Instrument Name: | Agilent 5975C |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | 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). |
| Ion Mode: | POSITIVE |
| Helium Flow: | 1 mL/min |
| Ionization Energy: | -70 eV |
