Return to study ST001480 main page
MB Sample ID: SA125017
| Local Sample ID: | Nl_32ppt4C_2 |
| Subject ID: | SU001554 |
| Subject Type: | Other |
| Subject Species: | Nitzschia lecointei;Fragilariopsis cylindrus;Navicula cf. perminuta;Navicula pelliculosa |
| Taxonomy ID: | 186028;186039;908978;913975 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001554 |
| Subject Type: | Other |
| Subject Species: | Nitzschia lecointei;Fragilariopsis cylindrus;Navicula cf. perminuta;Navicula pelliculosa |
| Taxonomy ID: | 186028;186039;908978;913975 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Nl_32ppt4C_2 | SA125017 | FL015329 | Nitzschia lecointei | Species |
| Nl_32ppt4C_2 | SA125017 | FL015329 | 32 | Salinity |
| Nl_32ppt4C_2 | SA125017 | FL015329 | 4 | Temp_degC |
Collection:
| Collection ID: | CO001549 |
| Collection Summary: | Axenic cultures of three Antarctic sea-ice diatoms (N. lecointei, N. cf. perminuta, and F. cylindrus) and two temperate diatoms (T. pseudonana and N. pelliculosa) were chosen for study. Cells were harvested during exponential growth onto 47 mm 0.2 µm PTFE filters (Omnipore) using combusted glassware and gentle filtration and stored at –80 °C until extraction. For each biological replicate (n = 2 for Antarctic species, n = 3 for temperate species), two 35 mL cultures were harvested onto each filter . An un-inoculated media blank was prepared and treated in the same manner as samples. |
| Sample Type: | Cultured diatom cells |
| Storage Conditions: | Described in summary |
Treatment:
| Treatment ID: | TR001569 |
| Treatment Summary: | Antarctic species were grown at −1°C and a PAR irradiance of 45 𝜇mol photons m−2 s−1 (16:8 light:dark cycle) using cool white lights. Temperate species were grown at 13°C and a PAR irradiance of 120 𝜇mol photons m−-2 s−-1(12:12 light:dark cycle). In both cases, light was saturating. Cultures were grown in artificial seawater (ESAW, salinity 31, for Antarctic species and Instant Ocean, salinity ~35 for temperate species). Cobalamin (vitamin B12) was replete in all cultures. To explore the effect of growth conditions on metabolic profiles using non-metric dimensional scaling analysis, samples were included of N. lecointei grown at temperatures of −1 and 4˚C and salinities of 32 and 41. |
Sample Preparation:
| Sampleprep ID: | SP001562 |
| Sampleprep Summary: | Each sample was extracted using a modified Bligh-Dyer extraction. Briefly, filters were cut up and put into 15 mL teflon centrifuge tubes containing a mixture of 100 µm and 400 µm silica beads. Heavy isotope-labeled internal standards were added along with ~2 mL of cold aqueous solvent (50:50 methanol:water) and ~3 mL of cold organic solvent (dichloromethane). The samples were shaken on a FastPrep-24 Homogenizer for 30 seconds and chilled in a -20 °C freezer repeatedly for three cycles of bead-beating and a total of 30 minutes of chilling. The organic and aqueous layers were separated by spinning samples in a centrifuge at 4,300 rpm for 2 minutes at 4 °C. The aqueous layer was removed to a new glass centrifuge tube. The remaining organic fraction was rinsed three more times with additions of 1 to 2 mL of 50:50 methanol:water. All aqueous rinses were combined for each sample and dried down under N2 gas. The remaining organic layer was transferred into a clean glass centrifuge tube and the remaining bead beating tube was rinsed two more times with cold organic solvent. The combined organic rinses were centrifuged, transferred to a new tube, and dried under N2 gas. Dried aqueous fractions were re-dissolved in 380 µL of water. Dried organic fractions were re-dissolved in 380 µL of 1:1 water:acetonitrile. 20 µL of isotope-labeled injection standards in water were added to both fractions. An un-inoculated media blank was prepared and treated in the same manner as the samples. |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Bligh-Dyer |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN002456 | AN002457 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Waters Acquity I-Class | Waters Acquity I-Class |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | mM (intracellular concentration) | mM (intracellular concentration) |
Chromatography:
| Chromatography ID: | CH001799 |
| Chromatography Summary: | See attached summary. |
| Methods Filename: | CH_Ingalls_Lab_LC_Methods.txt MS_Ingalls_Lab_MS_Methods.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 30 |
| Flow Rate: | 0.15 mL/min |
| Solvent A: | 85% acetonitrile/15% water; 10 mM ammonium carbonate |
| Solvent B: | 15% acetonitrile/85% water; 10 mM ammonium carbonate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002276 |
| Analysis ID: | AN002456 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | MS_Ingalls_Lab_MS_Methods.txt |
| MS ID: | MS002277 |
| Analysis ID: | AN002457 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | MS_Ingalls_Lab_MS_Methods.txt |
