Return to study ST001721 main page
MB Sample ID: SA162038
| Local Sample ID: | FH5_2 |
| Subject ID: | SU001798 |
| Subject Type: | Invertebrate |
| Subject Species: | Cherax tenuimanus |
| Taxonomy ID: | 99755 |
| Weight Or Weight Range: | 116g-198g |
| Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001798 |
| Subject Type: | Invertebrate |
| Subject Species: | Cherax tenuimanus |
| Taxonomy ID: | 99755 |
| Weight Or Weight Range: | 116g-198g |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| FH5_2 | SA162038 | FL019157 | Female | Sex |
| FH5_2 | SA162038 | FL019157 | 2 | Timepoint |
Collection:
| Collection ID: | CO001791 |
| Collection Summary: | All haemolymph collections for all animals occurred at the same time of day and the animals were handled in the same order on each of the four collection dates. Haemolymph (200mL) was extracted from the ventral sinus of each crayfish using a 21G needle and 1mL syringe inserted into the soft tissue at the base of the 5th pereopod. Haemolymph was added to 2mL Eppendorf tubes preloaded with 600µL LC-MS grade acetonitrile (Optima, Thermo Fisher Scientific, AUS) containing deuterated internal standards (d8- valine, d9 –trimethylamine-N-oxide (TMAO) , d3-leucine, d6-trans-cinnamic acid, d5-tryptophan, 1g/mL) from Cambridge Isotope Laboratories (Cambridge, MA, USA) and stored on ice. Samples were mixed at 1400rpm (Thermal Mixer, Thermo Fisher Scientific, AUS) for 60 seconds at 4°C, then centrifuged (Heraeus Megafuge 8R, Thermo Fisher Scientific, AUS) for 20 minutes (1800 g) at 4°C. After centrifuging, 100µL of supernatant was aliquoted into five separate vials. The samples were then dried using a SpeedVac centrifugal vacuum concentrator (Thermo Fisher Scientific, AUS). The dried samples were stored at -80°C for subsequent metabolomics analysis. |
| Collection Protocol Filename: | EmilyLette_20210225_014138_PR_CO_Methods__Lette_marron.pdf |
| Sample Type: | Hemolymph |
| Collection Method: | aspiration |
| Collection Location: | ventral sinus accessed from soft tissue at base of 5th pereopod |
| Storage Conditions: | -80℃ |
| Collection Vials: | 2mL Eppendorf tubes |
| Storage Vials: | 2mL Eppendorf tubes |
Treatment:
| Treatment ID: | TR001811 |
| Treatment Summary: | Cherax tenuimanus (n=10) were housed in aquaria as individuals prior to sampling at timepoint 1. After timepoint 1 they were placed in pairs with a potential mate of the opposite sex and housed in aquaria over the next four weeks which included timepoints 2-4. |
Sample Preparation:
| Sampleprep ID: | SP001804 |
| Sampleprep Summary: | The dried haemolymph samples were reconstituted using 100µL of LC-MS water containing 0.1% Formic acid. Samples were manually swirled, then placed in a thermomixer for 2 minutes at 4oC, before being centrifuged at (1800 g) for 5 minutes at 4oC. Next, 40µL of the supernatant was transferred into LC-MS amber vials with inserts and placed in the autosampler kept at 6oC. The order in which samples were analysed was randomised to avoid any potential instrument bias. A pooled quality control (QC) sample was prepared by adding 40 µL from each reconstituted sample to a single Eppendorf tube, which was then mixed to homogenise in a thermal mixer and centrifuged as above. This pooled sample was aliquoted (40µL) into LC-MS amber vials to create 16 QC samples and placed into the autosampler tray kept at 6°C ready for analysis. Samples were analysed within 24 hours from preparation. At the start of the analytical batch, a solvent blank, matrix blank, and ten conditioning QC samples were analysed (Broadhurst et al., 2018). QC samples were then injected after every fifth marron haemolymph sample with two QCs analysed at the end of the batch, following the standard protocols for metabolomics (Broadhurst et al., 2018). |
| Processing Storage Conditions: | Described in summary |
Combined analysis:
| Analysis ID | AN002804 | AN002805 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | Thermo Hypersil GOLD C18 (100 x 2.1mm,1.9m) | Thermo Hypersil GOLD C18 (100 x 2.1mm,1.9m) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area values | peak area values |
Chromatography:
| Chromatography ID: | CH002072 |
| Methods Filename: | Methods_Lette_marron |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Thermo Hypersil GOLD C18 (100 x 2.1mm,1.9m) |
| Column Temperature: | 45 |
| Flow Gradient: | isocratic at 99% A: 1 min; 50% B:1-2 min; linear increase to 99% B: over 7min; 99% B: 2min; initial conditions returned over 2 min; 100% A: 3 min. |
| Flow Rate: | 0.3mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002599 |
| Analysis ID: | AN002804 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Full scans with data-dependent tandem mass spectrometry were acquired on the Orbitrap mass analyzer. Full scans were acquired at a resolution of 70,000 at mass-to-charge ratio (m/z) 200 over the m/z range 70–1000 with the ESI conditions as follows: source heater = 350°C, sheath gas = 35 (arbitrary units), auxiliary gas = 10 (arbitrary units), capillary temperature 350°C, ion spray voltage = 3.0 kV (positive ion mode) and 2.5 kV (negative ion mode), S-lens 50%, and automatic gain control = 110¬¬¬-6. Tandem mass spectrometry experiments were performed at a resolution of 17,500 at m/z 200 on each sample with the higher energy collisional dissociation energy set at 20 eV. Data acquisition was carried out using Xcalibur software (Thermo Fisher Scientific). Before analysis, the Orbitrap was externally calibrated using ready-made calibration solutions (ESI-negative ion calibration and ESI-positive ion calibration solutions) obtained from Thermo Fisher Scientific. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Methods_Lette_marron |
| MS ID: | MS002600 |
| Analysis ID: | AN002805 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Full scans with data-dependent tandem mass spectrometry were acquired on the Orbitrap mass analyzer. Full scans were acquired at a resolution of 70,000 at mass-to-charge ratio (m/z) 200 over the m/z range 70–1000 with the ESI conditions as follows: source heater = 350°C, sheath gas = 35 (arbitrary units), auxiliary gas = 10 (arbitrary units), capillary temperature 350°C, ion spray voltage = 3.0 kV (positive ion mode) and 2.5 kV (negative ion mode), S-lens 50%, and automatic gain control = 110¬¬¬-6. Tandem mass spectrometry experiments were performed at a resolution of 17,500 at m/z 200 on each sample with the higher energy collisional dissociation energy set at 20 eV. Data acquisition was carried out using Xcalibur software (Thermo Fisher Scientific). Before analysis, the Orbitrap was externally calibrated using ready-made calibration solutions (ESI-negative ion calibration and ESI-positive ion calibration solutions) obtained from Thermo Fisher Scientific. |
| Ion Mode: | NEGATIVE |
