Return to study ST001807 main page
MB Sample ID: SA167810
| Local Sample ID: | Dm_polpos_QC_1 |
| Subject ID: | SU001884 |
| Subject Type: | Invertebrate |
| Subject Species: | Daphnia magna |
| Taxonomy ID: | 35525 |
| Age Or Age Range: | neonates < 9 h old |
Select appropriate tab below to view additional metadata details:
Sample Preparation:
| Sampleprep ID: | SP001890 |
| Sampleprep Summary: | all solvents were chilled to 4 ºC. A mixture of 320 µL of HPLC grade MeOH and 128 µL of HPLC grade H2O were added to each sample tube and kept on ice. Tubes were then placed in a Precellys 24 homogeniser for 2 × 10s bursts at 6400 rpm. The homogenised mixture was then transferred into a clean 1.8 mL glass vial (Fisher TUL 520 006 J) using a Pasteur pipette. 320 µL (32 µL/mg) CHCl3 (HPLC grade) and 160 µL (16 µL/mg) dH2O (HPLC grade) were then added to each vial. These vials were vortexed on full power for 15 s each to thoroughly mix polar and non-polar solvents. Vials were then left on ice for on ice for 10 min to allow initial phase separation. Vials were then centrifuged at 4000 rpm at 4 ºC for 10 min to ensure complete phase separation. Centrifuged vials were allowed to come to room temperature by setting them on the lab bench for 5 min. Samples were then visibly biphasic, with protein debris separating the upper (polar) and lower (non-polar) layers. A 500 uL Hamilton syringe was then used to remove the polar phase (~ 2 × 150 µL aliquots) into 2 clean 1.5 mL Eppendorf tubes (one for positive, one for negative ion analysis). Polar samples were then dried using a Speed Vac concentrator and stored at -80 °C until analysis. Sample Preparation and Direct Infusion Mass Spectrometry Metabolomics For positive ion analysis, 30 µL of 4 ºC 80:20 methanol:water plus 0.25% formic acid was added to each of the frozen, dried extracts, and each sample vortexed for 30s. For negative ion analysis 30 µL of 4 ºC 80:20 methanol:water plus 20 mM ammonium acetate was added to each of the frozen, dried extracts, and each sample vortexed for 30 s. Samples were then centrifuged at 4000 g at 4 ºC for 10 mins. For both positive and negative ion analyses, samples were randomized and 5 µL of sample supernatant was pipetted into a pre-washed 96-well sample plate in quadruplicate. Three quality control (QC) samples (a mixture with equal volume from all samples) and a blank were also included on each plate. Loaded plates were covered with a foil seal using heat sealer and loaded into a TriVersa Nano-Mate® nanoelectro-spray ion source (Advion) with the cooler set at 10 ºC. Non-targeted analysis was carried out on polar fractions by direct infusion mass spectrometry (DIMS) using an LTQ Orbitrap Elite (Thermo Fisher Scientific). 21 overlapping selected ion monitoring (SIM) windows were collected covering m/z values from 50 to 620. |
| Processing Storage Conditions: | 4℃ |
| Extract Storage: | 4℃ |
