Return to study ST001900 main page
MB Sample ID: SA176179
| Local Sample ID: | MP_Blank_01 |
| Subject ID: | SU001978 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J, C57BL/6J.rag1−/− |
| Age Or Age Range: | 6-8 weeks |
| Gender: | Female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001978 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J, C57BL/6J.rag1−/− |
| Age Or Age Range: | 6-8 weeks |
| Gender: | Female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| MP_Blank_01 | SA176179 | FL021854 | Blank | Genotype |
| MP_Blank_01 | SA176179 | FL021854 | Blank | Treatment |
Collection:
| Collection ID: | CO001971 |
| Collection Summary: | Two independent experiments were conducted, each with 6 mice per treatment group (30 individuals in total). Mice were euthanized by CO2 asphyxiation and their blood was taken by cardiac puncture into lithium-heparin coated tubes. Samples were spun for 5 min at 5000 rpm (approx. 7,043 × g) and plasma was immediately aliquoted into 1.5 mL tubes. In addition, a global sample pool containing equal volumes of each sample was prepared as quality control (QC) and four aliquots were transferred into 1.5 mL tubes. Finally, collection tube blank extractions were performed in triplicate by adding 1x PBS (same volume as blood collection) to lithium-heparin tubes and then transferring an aliquot into a 1.5 mL tube |
| Sample Type: | Blood (plasma) |
Treatment:
| Treatment ID: | TR001990 |
| Treatment Summary: | Genotype: Treatment C57BL/6J Naïve: Control, C57BL/6J mice were intraperitoneally injected (200 uL) with saline (0.9%) 9 hours prior to plasma acquisition. C57BL/6J Acute: Infected, C57BL/6J mice were infected with 10^5 Plasmodium berghei ANKA parasitised red blood cells 5 days prior to plasma acquisition and intraperitoneally injected (200 uL) with saline (0.9%) 9 hours prior to plasma acquisition for analysis. C57BL/6J LPS: LPS treatment, C57BL/6J mice were intraperitoneally injected (200 uL) with lipopolysaccharides (LPS) (0.75 mg/mL), from E.coli O127:B8, 9 hours prior to plasma acquisition. rag1-/- Naïve: Immune deficient control, C57BL/6J.rag1-/- mice were intraperitoneally injected (200 uL) with saline (0.9%) 9 hours prior to plasma acquisition. rag1-/- Acute: Immune deficient infected control, C57BL/6J.rag1-/- mice were infected with 10^5 Plasmodium berghei ANKA parasitised red blood cells 5 days prior to plasma acquisition and intraperitoneally injected (200 uL) with saline 9 hours prior to plasma acquisition for analysis. |
Sample Preparation:
| Sampleprep ID: | SP001984 |
| Sampleprep Summary: | Ten-times the sample volume of ice-cold butanol/methanol (1:1) containing 50 µg/mL antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT) was added to each sample and vortexed for 10 s. Samples were snap frozen and transported on dry ice. Subsequently, samples were thawed on ice and labelled in a randomized order. Samples were sonicated for 15 min in an ice-cold water bath sonicator, stored for 2 hrs at -30oC and then centrifuged for 15 min at 16,000 × g (4oC). Lastly, samples were aliquoted, dried down using a vacuum concentrator and stored at -80oC until LC/MS analysis |
Combined analysis:
| Analysis ID | AN003087 | AN003088 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Agilent 1290 Infinity II | Agilent 1290 Infinity II |
| Column | Agilent Zorbax HILIC Plus RRHD (100 x 2.1mm,1.8um,95Å) | Agilent Zorbax HILIC Plus RRHD (100 x 2.1mm,1.8um,95Å) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Agilent 6545 QTOF | Agilent 6545 QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002279 |
| Chromatography Summary: | Metabolite separation was performed on a Zorbax HILIC Plus RRHD (95Å, 1.8 µm, 2.1x100mm) analytical column connected to a 3 x 5 mm Zorbax HILIC Plus UHPLC guard column. The autosampler and column temperature were set to 4°C and 40°C, respectively. In positive and negative mode, eluent A was 10 mM ammonium acetate (pH neutral) in acetonitrile/milliQ water (95:5, v/v) and eluent B was 10 mM ammonium acetate (pH neutral) in acetonitrile/milliQ water (50:50, v/v). Total method runtime was 12 min with the following gradient for both modes: 0 min (1% eluent B) - 3.5 min (50% B) - 5.5 min (99%B) - 6.5 min (99% B) - 6.7 min (1% B) - 12 min (1% B). Flow rate was set to 0.5 mL/min. |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Agilent Zorbax HILIC Plus RRHD (100 x 2.1mm,1.8um,95Å) |
| Column Temperature: | 40 |
| Flow Gradient: | Total method runtime was 12 min with the following gradient for both modes: 0 min (1% eluent B) - 3.5 min (50% B) - 5.5 min (99%B) - 6.5 min (99% B) - 6.7 min (1% B) - 12 min (1% B). Flow rate was set to 0.5 mL/min. |
| Flow Rate: | 0.5 mL/min |
| Solvent A: | 95% acetonitrile/5% water; 10 mM ammonium acetate,pH neutral |
| Solvent B: | 50% acetonitrile/50% water; 10 mM ammonium acetate,pH neutral |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002869 |
| Analysis ID: | AN003087 |
| Instrument Name: | Agilent 6545 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | MS acquisition: The LC/MS platform consisted of a 1290 Infinity II UHPLC coupled to a 6545 QTOF mass spectrometer via Dual AJS ESI source (Agilent, Santa Clara, USA) and was controlled using MassHunter data acquisition software (v.10.1). Assessment of MS instrument performance and usage of reference ions were also performed as described previously. Full scan MS data (m/z 50-1700) was acquired at a scan rate of 2.5 spectra/sec (equals 3224 transients/spectrum) with the following source conditions: Gas temperature 250°C, gas flow 13 L/min, sheath gas temperature and flow at 400°C and 12 L/min, respectively, nebulizer 30 psi, fragmentor 135, capillary voltage at +4500 V and -4000 V, nozzle voltage was zero. Data processing: Positive and negative mode data was analysed separately. Data files (30 sample files, 6 QC files and 3 tube blank extraction files) were loaded into MassHunter Profinder (v 10 SP1, Agilent) and assigned to sample groups. Spectral feature extraction was performed using the recursive feature extraction method employing default settings with minor adjustments: Peak extraction was restricted to retention time (Rt) range 0-6.5 min, compound binning and alignment tolerances were set to 1% + 0.3 min for Rt and 20 ppm + 2 mDa for mass, integrator Agile 2 was used for peak integration, peak filters were set to at least 2500 counts and features must have satisfied filter conditions in at least 75 % of files in at least one sample group. Feature peak area was exported and data cleaning was performed using an in-house R script compiled of the following steps. Features were deleted if they: had a mean QC/tube blank area ratio of < 10; were absent across all QC samples; and had duplicates present. In addition, samples with a TIC scaling factor more than 50% above or below the median TIC were removed. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002870 |
| Analysis ID: | AN003088 |
| Instrument Name: | Agilent 6545 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | MS acquisition: The LC/MS platform consisted of a 1290 Infinity II UHPLC coupled to a 6545 QTOF mass spectrometer via Dual AJS ESI source (Agilent, Santa Clara, USA) and was controlled using MassHunter data acquisition software (v.10.1). Assessment of MS instrument performance and usage of reference ions were also performed as described previously. Full scan MS data (m/z 50-1700) was acquired at a scan rate of 2.5 spectra/sec (equals 3224 transients/spectrum) with the following source conditions: Gas temperature 250°C, gas flow 13 L/min, sheath gas temperature and flow at 400°C and 12 L/min, respectively, nebulizer 30 psi, fragmentor 135, capillary voltage at +4500 V and -4000 V, nozzle voltage was zero. Data processing: Positive and negative mode data was analysed separately. Data files (30 sample files, 6 QC files and 3 tube blank extraction files) were loaded into MassHunter Profinder (v 10 SP1, Agilent) and assigned to sample groups. Spectral feature extraction was performed using the recursive feature extraction method employing default settings with minor adjustments: Peak extraction was restricted to retention time (Rt) range 0-6.5 min, compound binning and alignment tolerances were set to 1% + 0.3 min for Rt and 20 ppm + 2 mDa for mass, integrator Agile 2 was used for peak integration, peak filters were set to at least 2500 counts and features must have satisfied filter conditions in at least 75 % of files in at least one sample group. Feature peak area was exported and data cleaning was performed using an in-house R script compiled of the following steps. Features were deleted if they: had a mean QC/tube blank area ratio of < 10; were absent across all QC samples; and had duplicates present. In addition, samples with a TIC scaling factor more than 50% above or below the median TIC were removed. |
| Ion Mode: | NEGATIVE |
