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MB Sample ID: SA209273
| Local Sample ID: | Hgd82.3 - No treatment NEG |
| Subject ID: | SU002265 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002265 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Hgd82.3 - No treatment NEG | SA209273 | FL025536 | No Treatment (control) | Treatment group |
Collection:
| Collection ID: | CO002258 |
| Collection Summary: | A murine model of AKU was used for all experiments as described previously (Preston et al., 2014). Seventeen BALB/c Hgd-/- mice (Figure 5 for age and gender) were included in this study, 8 mice were administered nitisinone (4 mg/L) through their drinking water for 6 days, the remaining 9 mice received no nitisinone. All animals were housed in air conditioned rooms (with a 12 h dark/light cycle) at 20 °C and 53 % humidity, with access to food and water ad libitum at Liverpool John Moores University. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. All mice were culled by carbon dioxide asphyxiation and CSF was removed from the cisterna magna following the puncture technique of Liu et al. (56), with some modifications. The cisterna magna was exposed by dissecting skin and overlaying muscles. Cauterisation was used to dry up any bleeding from surrounding tissues. A pulled glass pipette with an internal diameter of ~0.4 mm was attached to silicone tubing and connected to a 1 mL syringe with a 19 g needle. The tip of the glass pipette was used to puncture the membrane and held still just below the membrane by 1 person. Through a double-headed microscope a second person then used the 1 mL syringe to apply gentle pressure to encourage the CSF to flow up the capillary tube. Only clear CSF samples (i.e. non blood stained) were collected. Once collected CSF samples were transferred to clear Eppendorf tubes and stored at -80 °C until analysis. Samples were not acidified. The same glass pipette was used to collect CSF from each animal, and was washed with pure water between each animal. |
| Sample Type: | CSF |
Treatment:
| Treatment ID: | TR002277 |
| Treatment Summary: | 9 mice control 8 mice received 4 mg/L nitisinone for 6 days in drinking water |
Sample Preparation:
| Sampleprep ID: | SP002271 |
| Sampleprep Summary: | Murine CSF samples were prepared by diluting 2 µL CSF with 2 µL deionized water (DIRECT-Q 3UV Millipore water purification system). CSF samples were pipetted directly into a 150 µL glass insert with polymer feet, which sat in a 2 mL amber screw vial (Agilent, Cheadle, UK). These vials were used to ensure maximal recovery of sample. Deionised water was also added directly to the glass insert, and mixed with the CSF sample using a pipette. The glass vials were centrifuged at 600 rpm for 10 min to ensure the diluted sample was at the bottom of the vial for sampling. 1 µL of diluted CSF was analysed in negative and positive polarities, respectively. |
Combined analysis:
| Analysis ID | AN003568 | AN003569 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Agilent Infinity II | Agilent Infinity II |
| Column | Atlantis dC18 (3.0x100mm,3m,Waters,UK) | Atlantis dC18 (3.0x100mm,3m,Waters,UK) |
| MS Type | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole |
| MS instrument name | Agilent 6550 QTOF | Agilent 6550 QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | area | area |
Chromatography:
| Chromatography ID: | CH002639 |
| Chromatography Summary: | Liquid chromatography (LC) was performed on an Agilent 1290 Infinity II LC system. An Atlantis dC18 column (3.0x100mm, 3µm, Waters, UK) was maintained at 60°C with a flow rate of 0.4mL/min. Mobile phases were (A) water and (B) methanol both containing 5mmol/L ammonium formate and 0.1% formic acid. The elution gradient started at 5% mobile phase B at 0-1 min increasing linearly to 100% B by 12 min, held at 100% B until 14 min, returning to 95% A for 5 min to recondition the column. Injection volume was 1µL. |
| Instrument Name: | Agilent Infinity II |
| Column Name: | Atlantis dC18 (3.0x100mm,3m,Waters,UK) |
| Column Temperature: | 60 |
| Flow Gradient: | The elution gradient started at 5% mobile phase B at 0-1 min increasing linearly to 100% B by 12 min, held at 100% B until 14 min, returning to 95% A for 5 min to recondition the column |
| Flow Rate: | 0.4mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 5 mM ammonium formate |
| Solvent B: | 100% acetonitrile; 0.1% formic acid;5 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003325 |
| Analysis ID: | AN003568 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Quadrupole time-of-flight mass spectrometry (QTOF-MS) conditions An Agilent 6550 QTOF-MS equipped with a dual jet stream electrospray ionisation source was operated in 2GHz mode, over the mass range of 50-1700, in negative and positive polarities. A reference mass correction solution was continually infused at a flow rate of 0.5 mL/min via an external isocratic pump (Agilent, UK) for constant mass correction (see Preparation of reference mass correction solution). Capillary and fragmentor voltages were 4000 V and 380 V, respectively. Desolvation gas temperature was 200 °C with flow rate at 15 L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min and nebulizer pressure was 40 psi and nozzle voltage 1000 V. Data acquisition rate was 3 spectra/s. Preparation of reference mass correction solution Reference mass correction solution was prepared in 95:5 methanol:water containing 5 mmol/L purine (CAS No. 120-73-0), 100 mmol/L trifluoroacetic acid ammonium salt (TFA, CAS No. 3336-58-1) and 2.5 mmol/L hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine (HP-0921, CAS No. 58943-98-9) (Agilent, Cheadle, UK). Reference ions monitored were: purine (m/z 121.0509) and HP-0921 (m/z 922.0098) (positive polarity) and TFA (m/z 112.9856), purine (m/z 119.0363) and HP-0921 (HP-0921 + formate adduct: m/z 966.0007) (negative polarity). Data acquisition and handling parameters Data were acquired using Acquisition (Build 06.00, Agilent, Cheadle, UK). Quality checks and processing of raw data files (Agilent ‘.d’ files) were performed with Qualitative Analysis software (Build 07.00, Agilent, Cheadle, UK). Extracted ion chromatograms of reference masses were performed to check mass accuracy remained <5 ppm throughout the run and that the reference ion signal did not drop out during the chromatographic run. In addition, to check chromatographic reproducibility binary pump pressure curves for injections across each analytical sequence were overlaid. Mass accuracy and chromatographic reproducibility were acceptable for all experiments performed. Acquired profiling sample data were mined for signals against an established in-house AMRT database of compounds (contains theoretical accurate mass, measured retention time, and empirical formula for 469 intermediary metabolites, MW 72-785) using ‘targeted feature extraction’ with Profinder software (Build 08.00, Agilent, Cheadle, UK). Targeted feature extraction uses the molecular formulae from the AMRT database to extract and group spectral signals (i.e. adducts, isotopes and multimers) that correspond to individual database compounds. Feature extraction employed a window of theoretical accurate mass ±10 ppm and database retention time ±0.3 min. Allowed species were: H+, Na+ and NH4+ (positive polarity) and H- and CHO2- (negative polarity). Dimers were allowed for both polarities. Charge state range was 1-2. Data files were then exported from Profinder as ‘.CEF’ files as a whole batch for each profiling experiment and imported to Mass Profiler Professional (MPP) software for statistical analysis (Build 14.5, Agilent, Cheadle, UK). |
| Ion Mode: | POSITIVE |
| MS ID: | MS003326 |
| Analysis ID: | AN003569 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Quadrupole time-of-flight mass spectrometry (QTOF-MS) conditions An Agilent 6550 QTOF-MS equipped with a dual jet stream electrospray ionisation source was operated in 2GHz mode, over the mass range of 50-1700, in negative and positive polarities. A reference mass correction solution was continually infused at a flow rate of 0.5 mL/min via an external isocratic pump (Agilent, UK) for constant mass correction (see Preparation of reference mass correction solution). Capillary and fragmentor voltages were 4000 V and 380 V, respectively. Desolvation gas temperature was 200 °C with flow rate at 15 L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min and nebulizer pressure was 40 psi and nozzle voltage 1000 V. Data acquisition rate was 3 spectra/s. Preparation of reference mass correction solution Reference mass correction solution was prepared in 95:5 methanol:water containing 5 mmol/L purine (CAS No. 120-73-0), 100 mmol/L trifluoroacetic acid ammonium salt (TFA, CAS No. 3336-58-1) and 2.5 mmol/L hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine (HP-0921, CAS No. 58943-98-9) (Agilent, Cheadle, UK). Reference ions monitored were: purine (m/z 121.0509) and HP-0921 (m/z 922.0098) (positive polarity) and TFA (m/z 112.9856), purine (m/z 119.0363) and HP-0921 (HP-0921 + formate adduct: m/z 966.0007) (negative polarity). Data acquisition and handling parameters Data were acquired using Acquisition (Build 06.00, Agilent, Cheadle, UK). Quality checks and processing of raw data files (Agilent ‘.d’ files) were performed with Qualitative Analysis software (Build 07.00, Agilent, Cheadle, UK). Extracted ion chromatograms of reference masses were performed to check mass accuracy remained <5 ppm throughout the run and that the reference ion signal did not drop out during the chromatographic run. In addition, to check chromatographic reproducibility binary pump pressure curves for injections across each analytical sequence were overlaid. Mass accuracy and chromatographic reproducibility were acceptable for all experiments performed. Acquired profiling sample data were mined for signals against an established in-house AMRT database of compounds (contains theoretical accurate mass, measured retention time, and empirical formula for 469 intermediary metabolites, MW 72-785) using ‘targeted feature extraction’ with Profinder software (Build 08.00, Agilent, Cheadle, UK). Targeted feature extraction uses the molecular formulae from the AMRT database to extract and group spectral signals (i.e. adducts, isotopes and multimers) that correspond to individual database compounds. Feature extraction employed a window of theoretical accurate mass ±10 ppm and database retention time ±0.3 min. Allowed species were: H+, Na+ and NH4+ (positive polarity) and H- and CHO2- (negative polarity). Dimers were allowed for both polarities. Charge state range was 1-2. Data files were then exported from Profinder as ‘.CEF’ files as a whole batch for each profiling experiment and imported to Mass Profiler Professional (MPP) software for statistical analysis (Build 14.5, Agilent, Cheadle, UK). |
| Ion Mode: | NEGATIVE |
