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MB Sample ID: SA127153

Local Sample ID:449_A
Subject ID:SU001588
Subject Type:Other
Subject Species:Marine Plankton
Gender:Not applicable

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Subject:

Subject ID:SU001588
Subject Type:Other
Subject Species:Marine Plankton
Gender:Not applicable

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
449_ASA127153FL015671Heterocapsa triquetraSpecies
449_ASA127153FL015671449Strain
449_ASA127153FL015671DinoflagellateBroad taxon

Collection:

Collection ID:CO001583
Collection Summary:Axenic phytoplankton were cultured in controlled laboratory settings and harvested under exponential growth using a gentle vacuum filtration onto 47 mm Durapore filters (pore size 0.2 µm). Samples were flash frozen in liquid N2 and stored at -80°C until extraction for metabolites.
Sample Type:Cultured plankton cells
Storage Conditions:Described in summary

Treatment:

Treatment ID:TR001603
Treatment Summary:Media, light, and temperature were chosen for optimal growth of each phytoplankton species. In short, axenic phytoplankton were cultured in controlled laboratory settings and harvested under exponential growth using a gentle vacuum filtration onto 47 mm Durapore filters (pore size 0.2 µm). Samples were flash frozen in liquid N2 and stored at -80°C until extraction. In addition to samples, media blanks corresponding to each media type were harvested and served as a matrix blank to each corresponding phytoplankton sample.

Sample Preparation:

Sampleprep ID:SP001596
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 cold 50:50 methanol:water. All aqueous rinses were combined for each sample and ~2mL of cold dichloromethane was added to the combined aqueous layer. Tubes were shaken and centrifuged at 4,300 rpm for 2 minutes at 4°C. The aqueous layer was removed to a new glass vial and dried under N2 gas. The remaining organic layer in the bead beating tubes was transferred into the glass centrifuge tube and the bead beating tube was rinsed two more times with cold organic solvent. The combined organic rinses were centrifuged, transferred to a new glass vial, 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. Media blanks were extracted alongside samples as methodological blanks.
Processing Storage Conditions:On ice
Extraction Method:Bligh-Dyer
Extract Storage:-80℃

Combined analysis:

Analysis ID AN002508 AN002509 AN002510
Analysis type MS MS MS
Chromatography type Reversed phase HILIC HILIC
Chromatography system Waters Acquity I-Class Waters Acquity I-Class Waters Acquity I-Class
Column Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) SeQuant ZIC-pHILIC (150 x 2.1mm,5um) SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
MS Type ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE POSITIVE NEGATIVE
Units Adjusted and normalized peak areas Adjusted and normalized peak areas Adjusted and normalized peak areas

Chromatography:

Chromatography ID:CH001833
Chromatography Summary:See attached summary.
Methods Filename:Ingalls_Lab_LC_Methods.txt
Instrument Name:Waters Acquity I-Class
Column Name:Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um)
Column Temperature:35
Flow Rate:0.4 ml/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH001834
Chromatography Summary:See attached summary
Methods Filename:Ingalls_Lab_LC_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:MS002326
Analysis ID:AN002508
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:Ingalls_Lab_MS_Methods.txt
  
MS ID:MS002327
Analysis ID:AN002509
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:Ingalls_Lab_MS_Methods.txt
  
MS ID:MS002328
Analysis ID:AN002510
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:Ingalls_Lab_MS_Methods.txt
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