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MB Sample ID: SA213097
| Local Sample ID: | IgEki (OVA_EW) 1S |
| Subject ID: | SU002322 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002322 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| IgEki (OVA_EW) 1S | SA213097 | FL025985 | IgE knock-in mice | Genotype |
| IgEki (OVA_EW) 1S | SA213097 | FL025985 | Sensitised to ovalbumin | Ovalbumin sensitisation |
Collection:
| Collection ID: | CO002315 |
| Collection Summary: | Faecal and serum samples were obtained and stored at -80°C until metabolite extraction. |
| Sample Type: | Feces; Blood (serum) |
| Storage Conditions: | -80? |
Treatment:
| Treatment ID: | TR002334 |
| Treatment Summary: | Mice (female, 6 to 8 weeks old) were sensitized by an intraperitoneal injection with 10 µg of ovalbumin (OVA, Sigma-Aldrich) and 1 mg of Alum (ThermoFisher Scientific) in 500 µl of PBS or treated only with PBS twice as non-sensitized controls, at a two-week interval. |
Sample Preparation:
| Sampleprep ID: | SP002328 |
| Sampleprep Summary: | FAECES. Frozen faeces samples were lyophilized overnight at -80°C. Faecal pellets were doubly extracted (i) first with MeOH:MTBE (4:1, v/v) and (ii) with MeOH:H2O (4:1, v/v). Equal volumes (300 µL) of the supernatants from (i) and (ii) were then mixed and aliquoted for subsequent analysis. SERUM. Serum samples (100 µL) were deproteinized by an addition of 300 µL of cold acetonitrile (1:3 v/v) and homogenized for 15 min in a vortex. After a 10 min ice bath, samples were centrifuged (16000 x g, 4°C, 20 min) and stored at -20°C until the start of sample treatment. The resulting supernatants were filtered through 0.22 µm for LC-MS, 80 µL were transferred into an analytical vial for their analysis. For CE-MS, 80 µL of the supernatants were evaporated to dryness using a Speedvac Concentrator and reconstituted in 80 µL of MilliQ® water containing an internal standard (0.2 mM L-methionine sulfone) and 0.1 M FA. Finally, for GC-MS, 120 µL of the corresponding aliquots were evaporated to dryness using a Speedvac Concentrator, followed by the addition of 10 µL of O-methoxyamine hydrochloride (15 mg/mL) in pyridine for methoximation. After gently vortexing, the vials were incubated in darkness at room temperature for 16 h. Then, 10 µL of BSTFA with 1 % TMCS (v/v) were added and samples were vortexed for 5 min, silylation was carried out for 1 h at 70 °C and finally 120 µL of C18:0 methyl ester (10 mg/L in heptane) were added as an internal standard and samples were mixed again by gentle vortexing. Eight blank samples were prepared for GC-MS by the same procedure of extraction and derivatization. |
| Sampleprep Protocol Filename: | Protocolsamples.docx |
| Processing Method: | Lyophilization, homogenization, protein precipitation and metabolite extraction |
| Processing Storage Conditions: | On ice |
Combined analysis:
| Analysis ID | AN003646 | AN003647 | AN003648 | AN003649 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | CE | GC |
| Chromatography system | Agilent 1290 | Agilent 1290 | Agilent 7100 CE | Agilent 7890A |
| Column | Zorbax Extend C18 (50 x 2.1 mm,1.8um) | Zorbax Extend C18 (50 x 2.1 mm,1.8um) | Fused-silica capillary (total length,100 cm;,50um) | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| MS Type | ESI | ESI | ESI | EI |
| MS instrument type | QTOF | QTOF | TOF | Single quadrupole |
| MS instrument name | Agilent 6545 QTOF | Agilent 6545 QTOF | Agilent 6224 TOF | Agilent 5975C |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | POSITIVE |
| Units | Peak Area | peak area | Peak Area | Peak area |
Chromatography:
| Chromatography ID: | CH002700 |
| Chromatography Summary: | For LC-MS, 0.5 µL of sample were injected into a Zorbax Extend C18 (50 x 2.1 mm, 1.8 µm) maintained at 60 °C. The flow rate was set at 0.6 mL/min. . The flow rate was set at 0.6 mL/min. The elution gradient involved a mobile phase consisting of: (A) 0.1% formic acid (FA) in water and (B) 0.1% FA in acetonitrile (ACN). The gradient was 5% B (0–1 min), 5 to 80% B (1–7 min), 80 to 100% B (7–11.5 min), and 100 to 5% B (11.5–12 min). The system was finally held at 5% B for 3 min to re-equilibrate the system (15 min of total analysis time). The ESI data were acquired in both modes, ESI (+) and ESI (-), in separate runs. The capillary voltage was set at 3,000V for ESI (+) and 4,000V for ESI (-). The drying gas flow rate was 12 L/min at 250 °C and gas nebulizer at 52 psi; nozzle voltage was 1000V; fragmentor voltage was 175 V; skimmer and octopole radio frequency voltage (OCT RF Vpp) were set to 65 and 750 V, respectively. Data were collected in the centroid mode at a scan rate of 1.5 spectra per second. |
| Methods Filename: | Protocolmethods.docx |
| Instrument Name: | Agilent 1290 |
| Column Name: | Zorbax Extend C18 (50 x 2.1 mm,1.8um) |
| Column Temperature: | 60 |
| Flow Gradient: | 5% B (0-1 min), 5 to 80% B (1-7 min), 80 to 100% B (7-11.5 min), and 100 to 5% B (11.5-12 min). The system was finally held at 5% B for 3 min to re-equilibrate the system (15 min of total analysis time). |
| Flow Rate: | 0.6 mL/min |
| Sample Injection: | 0.5 µL |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002701 |
| Chromatography Summary: | For LC-MS, 0.5 µL of sample were injected into a Zorbax Extend C18 (50 x 2.1 mm, 1.8 µm) maintained at 60 °C. The flow rate was set at 0.6 mL/min. . The flow rate was set at 0.6 mL/min. The elution gradient involved a mobile phase consisting of: (A) 0.1% formic acid (FA) in water and (B) 0.1% FA in acetonitrile (ACN). The gradient was 5% B (0–1 min), 5 to 80% B (1–7 min), 80 to 100% B (7–11.5 min), and 100 to 5% B (11.5–12 min). The system was finally held at 5% B for 3 min to re-equilibrate the system (15 min of total analysis time). The ESI data were acquired in both modes, ESI (+) and ESI (-), in separate runs. The capillary voltage was set at 3,000V for ESI (+) and 4,000V for ESI (-). The drying gas flow rate was 12 L/min at 250 °C and gas nebulizer at 52 psi; nozzle voltage was 1000V; fragmentor voltage was 175 V; skimmer and octopole radio frequency voltage (OCT RF Vpp) were set to 65 and 750 V, respectively. Data were collected in the centroid mode at a scan rate of 1.5 spectra per second. |
| Methods Filename: | Protocolmethods.docx |
| Instrument Name: | Agilent 1290 |
| Column Name: | Zorbax Extend C18 (50 x 2.1 mm,1.8um) |
| Column Temperature: | 60 |
| Flow Gradient: | 5% B (0-1 min), 5 to 80% B (1-7 min), 80 to 100% B (7-11.5 min), and 100 to 5% B (11.5-12 min). The system was finally held at 5% B for 3 min to re-equilibrate the system (15 min of total analysis time). |
| Flow Rate: | 0.6 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002702 |
| Chromatography Summary: | For CE-MS, the separation occurred in a fused-silica capillary (Agilent: total length, 100 cm; i.d., 50 µm). All separations were performed in normal polarity with a background electrolyte containing 1.0 M formic acid in 10% MeOH (v/v) at 20°C. New capillaries were preconditioned with a flush of 1.0 M NaOH for 30 min, followed by MilliQ water for 30 min and the background electrolyte for 30 min. Before each analysis, the capillary was conditioned with a flush of background electrolyte for 5 min. The sheath liquid (10 µL/min) was MeOH:H2O (1:1) containing 1.0 mM FA with two reference masses 121.0509 (purine), and 922.0098 (HP), which allowed for correction and higher mass accuracy in the MS. The sheath liquid was infused by an ISO Pump (1200 Agilent). The samples were hydrodynamically injected at 50 mBar for 50 s. The stacking was performed by applying the background electrolyte at 100 mBar for 20 s. The separation voltage was 30 kV, the internal pressure was 25 mBar and the analyses were performed in 40 min. |
| Methods Filename: | Protocolmethods.docx |
| Instrument Name: | Agilent 7100 CE |
| Column Name: | Fused-silica capillary (total length,100 cm;,50um) |
| Chromatography Type: | CE |
| Chromatography ID: | CH002703 |
| Chromatography Summary: | For GC-MS, two microliters (2 µL) of the derivatized sample were injected through a DB5-MS GC-Column (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 (Helium) 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 Helium 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 °C (held for 1 min), increased to 325 °C at 10 °C/min rate (within 26.5 min) and was held at 325 °C for 10 min. The total run time was 37.5 min. A cool-down period of 10 min was applied before the next injection. |
| Methods Filename: | Protocolmethods.docx |
| Instrument Name: | Agilent 7890A |
| Column Name: | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| Chromatography Type: | GC |
MS:
| MS ID: | MS003397 |
| Analysis ID: | AN003646 |
| Instrument Name: | Agilent 6545 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | LC-MS analysis was performed on an Agilent HPLC system (1290 infinity II 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 (6545) system (Agilent Technologies, Waldbronn, Germany) |
| Ion Mode: | POSITIVE |
| MS ID: | MS003398 |
| Analysis ID: | AN003647 |
| Instrument Name: | Agilent 6545 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | LC-MS analysis was performed on an Agilent HPLC system (1290 infinity II 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 (6545) system (Agilent Technologies, Waldbronn, Germany) |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003399 |
| Analysis ID: | AN003648 |
| Instrument Name: | Agilent 6224 TOF |
| Instrument Type: | TOF |
| MS Type: | ESI |
| MS Comments: | CE-MS analysis was performed by a CE system (Agilent Technologies 7100) coupled to a time of flight analyzer (TOF; Agilent Technologies 6224) |
| Ion Mode: | POSITIVE |
| MS ID: | MS003400 |
| Analysis ID: | AN003649 |
| 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 |
