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Oxylipin and Lipid analysis

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» Sample list, experiments 1-5

» Sample list, experiments 6 & 7

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» Oxylipin Analysis raw data files

Fredricks / Van Mooy lab data files

Oxylipin / lipid analysis

General information - all analyses

Total lipid extract obtained following a modified Bligh & Dyer protocol. BHT (butylated hydroxyl toluene) added as an antioxidant, DNP-PE added as an internal std (dinitrophenylphosphatidylethanolamine, Avanti polar lipids).

Oxylipin analysis / lipidomics method.

Analyzed on Thermo Exactive plus Orbitrap mass spectrometer (electrospray ionization) coupled to an Agilent 1200 HPLC. Chromatography follows Hummel et al. (2011 Ultra Performance Liquid Chromatography and High Resolution Mass Spectrometry for the Analysis of Plant Lipids. Front. Plant Sci. 2: 54. Doi:10.3389/fpls.2011.00054).

Raw data is presented as .raw files from Xcalibur 2.2 software (Thermo). Conversion utilities are available on the web which will convert .raw files to a more accessible format like .mzXML

Polar membrane lipid analysis

Quantitative: “big 9” lipids on Thermo TSQ Vantage triple quadrupole mass spectrometer. Electrospray ionization, chromatography – normal phase on a diol column as published in Lipids:

Reference: Popendorf, K.J., Fredricks, H.F., and Van Mooy, B.A.S. (2013) Molecular-ion independent quantitation of intact polar diacylglycerolipids in marine plankton using triple quadrupole MS. Lipids 48: 185-195; doi:10.1007/s11745-012-3748-0.

Qualitative: Samples run on Thermo LCQ Fleet. An ion-trap mass spectrometer that provides low-resolution MS and MS/SM data and both positive and negative ion modes. HPLC as above.

Reference: Van Mooy, B.A.S., Fredricks, H.F. (2010). Bacterial and eukaryotic intact polar lipids in the eastern subtropical South Pacific: Water-column distribution, planktonic sources, and fatty acid composition. Geochimica et Cosmochimica Acta, 74 (22): 6499-6516; DOI: 10.1016/j.gca.2010.08.026.

Data

Data custodians:

Experiments 1-5 Helen Fredricks (hfredricks@whoi.edu)

Experiments 6-7 Bethanie Edwards (bedwards@whoi.edu)

Ftp link to raw data files: ftp://ftp.whoi.edu/pub/science/MCG/gbmf/VanMooy/OxylipinAnalysis

Sample list, experiments 1-5 (Excel file)
Sample list, experiments 6 & 7 (Excel file)

1.  Diatom cultures exposed to H2O2
Oxylipin analysis: Samples from: Kim Thamatrakoln / Kay Bidle (Rutgers), extracted and analyzed by Helen Fredricks. 5 cultures: T pseudonana, C socialis, C tenuissimus 2-10, C tenuissimus 2-6, C lorenzianus: Each exposed to 0, 30, 150 μM H2O2

2.  Pheaodactylum tricornutum culture exposed to H2O2
Oxylipin analysis: Samples from Assaf Vardi group, Weizmann Institute of Plant Sciences, extracted by Jeremy Tagliaferre, analyzed and processed by Helen Fredricks.

Phaeodactylum tricornutum culture exposed to H2O2 at 0, 30 and 150 uM concentrations, samples collected at 4, 8 and 24 hour intervals. In duplicate.

3.  Chaetoceros infected with DNA and RNA viruses
Lipid analysis: Samples from: Kim Thamatrakoln / Kay Bidle (Rutgers), extracted by Jeremy Tagliaferre, analyzed and processed by Helen Fredricks. All samples were analyzed on the LCQ, a single replicate from each day/treatment was analysed on the Exactive to give high res. data.

Chaetoceros strains 2-6 and 2-10 exposed to a DNA and a RNA virus. Samples for lipid analysis taken at ~ 1, 3, 5, 7 days. More details in the “Chaetoceros” sheets of the ‘sample list’ excel file.

4.  Emiliania Huxleyi strains grown at different calcium concentrations
Lipid analysis. Samples from Chris Johns / Kay Bidle, Rutgers. Samples extracted by Jeremy Taglaferre / Oliver Newman analysed and processed by Helen Fredricks.

Emiliania huxleyi strains 374, 607, 611, 624 and 659 grown at 0.1 and 10 mMol calcium. LCQ / ion trap data files provide low res. data.

5.  Viral infection of Emiliania huxleyi
A post-doc in Rutger’s Bidle lab group, Jozef Nissimov has made a couple of visits to the Van Mooy lab to extract and run samples. Samples analyzed by Helen Fredricks and Jozef Nissimov. LCQ ion trap samples provide low resolution MS and MS/MS data in positive and negative ion modes.

6.  PS1312- Research cruise in June/July 2013 in the upwelling region of coastal California
a.    Water Column Oxylipin Distribution
Particulate Lipid samples were collected by filtering 1L of seawater from 6 depth at 8 stations onto a 0.2 um Durapore filter. Lipids were extracted from the filters back in the lab using a modified Bligh and Dyer (1959) protocol (Popendorf et al. 2013). DNP-PE was added as an internal standard during extraction. Samples for dissolved lipids were collected at 2 depths (Chl max and the 55% PAR) at 8 stations along the cruise track by pre-filtering SW through a 0.2 um sterivex filter to remove particulates. Benzaldehyde was added to the filtrate as an internal standard (10uM). Then, approximately 200ml of filtrate were extracted onto a solid phase extraction (SPE) cartridge (Waters HBL). SPE cartridges were stored at -80C until elution and mass spec analysis (Edwards, in prep). Samples were analyzed using a two different reverse phase HPLC  methods paired with high resolution, accurate mass (HRAM) data from a Thermo Q Exactive mass spectrometer (<2 ppm)  adapted from Hummel et al. (2011). For quantification of free fatty acids, oxylipins, and intact polar lipid, electrospray ionization-HRAM was paired with a reverse phase chromatographic method using a C8 column (Agilent) as the stationary phase and a starting gradient of 55% water 45% 70:30 ACN:IPA increasing to 1% water 99% 70:30 ACN:IPA over 26 minutes with a 5 min equilibrations period. To optimize for the quantification of polyunsaturated aldehydes, atmospheric pressure chemical ionization-HRAM was paired with a reverse phase chromatographic method using a C18 column (Agilent) as the stationary phase and starting gradient of 80% water 20% 70:30 Methanol:IPA increasing to 1% water 99% 70:30 Methanol:IPA over 26 minutes with a 10 min equilibration period.

b.    Oxylipin Distributions in Sinking Particles
From the four traps deployed over 6-12 hours at 50m depth, particulate lipid samples were collected by filtering one-500 ml split of trap material onto a 0.2um Durapore filter. Lipids were extracted from the filters back in the lab using a modified Bligh and Dyer protocol (Popendorf et al. 2013). DNP-PE was added as an internal standard during extraction. Samples for dissolved lipids were collected from the four net traps as well by pre-filtering SW through a 0.2 um sterivex filter to remove particulates. Benzaldehyde was added to the filtrate as an internal standard (10uM). Then, approximately 200ml of filtrate were extracted onto a solid phase extraction (SPE) cartridge (Waters HBL). SPE cartridges were stored at -80C until elution and mass spec analysis (Edwards, in prep). Samples were analyzed using a two different reverse phase HPLC  methods paired with high resolution, accurate mass (HRAM) data from a Thermo Q Exactive mass spectrometer (<2 ppm)  adapted from Hummel et al. (2011). For quantification of free fatty acids, oxylipins, and intact polar lipid, electrospray ionization-HRAM was paired with a reverse phase chromatographic method using a C8 column (Agilent) as the stationary phase and a starting gradient of 55% water 45% 70:30 ACN:IPA increasing to 1% water 99% 70:30 ACN:IPA over 26 minutes with a 5 min equilibrations period. To optimize for the quantification of polyunsaturated aldehydes, atmospheric pressure chemical ionization-HRAM was paired with a reverse phase chromatographic method using a C18 column (Agilent) as the stationary phase and starting gradient of 80% water 20% 70:30 Methanol:IPA increasing to 1% water 99% 70:30 Methanol:IPA over 26 minutes with a 10 min equilibration period.

c.    Oxylipin Incubation Experiments
On-deck incubation experiments were conducted by incubating 20L of whole seawater collected from the 55% PAR depth horizon in the presence of various oxylipin compounds at a range of concentrations in triplicate (see Table ). The purpose of the experiment was to determine the response of natural surface ocean free-living microbial communities to various oxylipins and oxylipin concentrations. After 24 hours of incubation at 55% PAR and in situ temperature the triplicate incubations were harvested. Samples were collected for enzyme activity, particulate and dissolved lipidome, biomass, BSi, dSi, SRP, silicification rates, and viral counts.

Experiment Treatment # Treatment
I1 1-3 Control
I1 4-6 0.1 µM PUA
I1 7-9 1 µM PUA
I1 10-12 10 µM PUA
I2 1-3 Control
I2 4-6 0.1 µM ARA mix
I2 7-9 1 µM ARA mix
I2 10-12 10 µM ARA mix
I3 1-3 Control
I3 4-6 0.1 µM ARA mix
I3 7-9 1 µM ARA mix
PUA= heptadienal, octadienal, and decadienal
ARA mix= 70% Arachidonic acid, 30% hydroperoxy-eicosatetraenoic acid

Particulate Lipid samples were collected by filtering 1L of seawater from each triplicate onto a 0.2 um Durapore filter. Lipids were extracted from the filters back in the lab using a modified Bligh and Dyer (1959) protocol (Popendorf et al. 2013). DNP-PE was added as an internal standard during extraction. Samples for dissolved lipids were collected by  pre-filtering 1 L of each triplicate through a 0.2 um sterivex filter to remove particulates. Benzaldehyde was added to the filtrate as an internal standard (10uM). Then, approximately 200ml of filtrate were extracted onto a solid phase extraction (SPE) cartridge (Waters HBL). SPE cartridges were stored at -80C until elution and mass spec analysis (Edwards, in prep). Samples were analyzed using a two different reverse phase HPLC  methods paired with high resolution, accurate mass (HRAM) data from a Thermo Q Exactive mass spectrometer (<2 ppm)  adapted from Hummel et al. (2011). For quantification of free fatty acids, oxylipins, and intact polar lipid, electrospray ionization-HRAM was paired with a reverse phase chromatographic method using a C8 column (Agilent) as the stationary phase and a starting gradient of 55% water 45% 70:30 ACN:IPA increasing to 1% water 99% 70:30 ACN:IPA over 26 minutes with a 5 min equilibrations period. To optimize for the quantification of polyunsaturated aldehydes, atmospheric pressure chemical ionization-HRAM was paired with a reverse phase chromatographic method using a C18 column (Agilent) as the stationary phase and starting gradient of 80% water 20% 70:30 Methanol:IPA increasing to 1% water 99% 70:30 Methanol:IPA over 26 minutes with a 10 min equilibration period.

d.    Nutrient Amendment experiment
An on-deck nutrient amendment incubation experiment was conducted to determine how the oxylipin profile of natural phytoplankton populations changes under nutrient stress. Whole seawater was collected from the 55% PAR depth horizon and incubated in triplicate under three different conditions: Control (ambient nutrient stress), +NP (simulated Si stress), and +NPSi (replete). After a 72 hour incubation period, samples were collected for the dissolved and particulate lipidome, biomass, enzyme activity, and nutrients.

Treatment # Treatment Nutrient State
N1-N3 Control Ambient nutrient stress
N4-N6 +NP Stimulated Si stress
N7-N9 +NPSi Replete

Particulate Lipid samples were collected by filtering 1L of seawater from each triplicate onto a 0.2 um Durapore filter. Lipids were extracted from the filters back in the lab using a modified Bligh and Dyer (1959) protocol (Popendorf et al. 2013). DNP-PE was added as an internal standard during extraction. Samples for dissolved lipids were collected by  pre-filtering 1 L of each triplicate through a 0.2 um sterivex filter to remove particulates. Benzaldehyde was added to the filtrate as an internal standard (10uM). Then, approximately 200ml of filtrate were extracted onto a solid phase extraction (SPE) cartridge (Waters HBL). SPE cartridges were stored at -80C until elution and mass spec analysis (Edwards, in prep). Samples were analyzed using a two different reverse phase HPLC  methods paired with high resolution, accurate mass (HRAM) data from a Thermo Q Exactive mass spectrometer (<2 ppm)  adapted from Hummel et al. (2011). For quantification of free fatty acids, oxylipins, and intact polar lipid, electrospray ionization-HRAM was paired with a reverse phase chromatographic method using a C8 column (Agilent) as the stationary phase and a starting gradient of 55% water 45% 70:30 ACN:IPA increasing to 1% water 99% 70:30 ACN:IPA over 26 minutes with a 5 min equilibrations period. To optimize for the quantification of polyunsaturated aldehydes, atmospheric pressure chemical ionization-HRAM was paired with a reverse phase chromatographic method using a C18 column (Agilent) as the stationary phase and starting gradient of 80% water 20% 70:30 Methanol:IPA increasing to 1% water 99% 70:30 Methanol:IPA over 26 minutes with a 10 min equilibration period.

7.  Collaborations with Matt Johnson
a.    Impacts of growth phase and Si stress on the lipidome of diatom cultures
Culture experiments were conducted to determine how the lipidome of various diatoms shifts with growth phase and Si stress. The particulate and dissolved lipidomes were sampled from cultures of three diatoms isolated from the PS1312 (MJSUR 06, MJSUR12, MJSUR15) and three model diatom species (Chaetoceros socialis, Phaeodactylum tricornutum and Thalassiosira pseudonana) during exponential growth, stationary phase, and Si-limitation (except Pt which does not have a Si requirement).

Particulate Lipid samples were collected by filtering 200 mL of seawater from each triplicate onto a 0.2 um Durapore filter. Lipids were extracted from the filters back in the lab using a modified Bligh and Dyer (1959) protocol (Popendorf et al. 2013). DNP-PE was added as an internal standard during extraction. Samples for dissolved lipids were collected by  pre-filtering 200 mL of each triplicate through a 0.2 um sterivex filter to remove particulates. Benzaldehyde was added to the filtrate as an internal standard (10uM). Then, the 200ml filtrate was extracted onto a solid phase extraction (SPE) cartridge (Waters HBL). SPE cartridges were stored at -80C until elution and mass spec analysis (Edwards, in prep). Samples were analyzed using a two different reverse phase HPLC  methods paired with high resolution, accurate mass (HRAM) data from a Thermo Q Exactive mass spectrometer (<2 ppm)  adapted from Hummel et al. (2011). For quantification of free fatty acids, oxylipins, and intact polar lipid, electrospray ionization-HRAM was paired with a reverse phase chromatographic method using a C8 column (Agilent) as the stationary phase and a starting gradient of 55% water 45% 70:30 ACN:IPA increasing to 1% water 99% 70:30 ACN:IPA over 26 minutes with a 5 min equilibrations period. To optimize for the quantification of polyunsaturated aldehydes, atmospheric pressure chemical ionization-HRAM was paired with a reverse phase chromatographic method using a C18 column (Agilent) as the stationary phase and starting gradient of 80% water 20% 70:30 Methanol:IPA increasing to 1% water 99% 70:30 Methanol:IPA over 26 minutes with a 10 min equilibration period.

b.    PtNOA grazing experiment
Samples were collected for complete lipidome analysis from WT Phaeodactylum tricornutum and PtNOA cultures with and without the grazer Oxhyrris marinaat t=2 hrs and t=final over a six hour grazing experiment. The purpose of the experiment was to determine how the lipidome changes under general stress vs. under grazing pressure. Particulate lipid samples were collected by filtering 200 ml of each treatment onto 0.2 µm Durapore filters. The filters were stored at -80˚C and extracted in the lab using a modified Bligh and Dyer (1959) lipid extraction protocol (Popendorf et al. 2013). DNP-PE was added as an internal standard during extraction. Samples for dissolved lipids were pre-filtered through a 0.2 um sterivex filter to remove particulates. Benzaldehyde was added to the filtrate as an internal standard (10uM). Dissolved lipids were then extracted onto a solid phase extraction cartridge (Waters HBL) and stored at -80C until elution and mass spec analysis (Edwards, in prep).  Samples were analyzed using a two different reverse phase HPLC  methods paired with high resolution, accurate mass (HRAM) data from a Thermo Q Exactive mass spectrometer (<2 ppm)  adapted from Hummel et al. (2011). For quantification of free fatty acids, oxylipins, and intact polar lipid, electrospray ionization-HRAM was paired with a reverse phase chromatographic method using a C8 column (Agilent) as the stationary phase and a starting gradient of 55% water 45% 70:30 ACN:IPA increasing to 1% water 99% 70:30 ACN:IPA over 26 minutes with a 5 min equilibrations period. To optimize for the quantification of polyunsaturated aldehydes, atmospheric pressure chemical ionization-HRAM was paired with a reverse phase chromatographic method using a C18 column (Agilent) as the stationary phase and starting gradient of 80% water 20% 70:30 Methanol:IPA increasing to 1% water 99% 70:30 Methanol:IPA over 26 minutes with a 10 min equilibration period.

 

Next Step: Lipidome Annotation

The particulate and dissolved lipidomes are now being annotated in the software package, Metabolic Analysis and Visualization ENgine (MAVEN,) that detects peaks and facilitates pseudo-targeted lipidomics analysis (Melamud et al., 2010; Collins et al. in prep). Identities are assigned to individual peaks by querying a lipid database that Helen Fredricks populated with IPLs, FA, oxylipins, pigments, and sterols. The ability to assign ID based on m/z hinges on the high mass resolution afforded to us by the Thermo Q Exactive mass spec.

Works cited:

Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can. J. Physiol. Pharmacol. 37:911–917.

Collins JR et al. in prep. Pseudo-targeted lipidomics analysis of cultured marine microbes and natural marine microbial communities.

Edwards BR et al. in prep. A Novel Method for the Extraction and Quantification of Particulate and Dissolved Oxylipins from Marine Environments.

Hummel J et al. (2011) Ultra Performance Liquid Chromatotraphy and High Resolution Mass Spectrometry for the Analysis of Plant Lipids. Front. Plant Sci. 2: 54. Doi:10.3389/fpls.2011.00054

Melamud E et al. (2010) Metabolomic Analysis and Visualization Engine for LC-MS Data. Anal. Chem. 82: 9818-9826. Doi: 10.1021/ac1021166

Popendorf KJ et al. (2013) Molecular Ion-Independent Quantification of Polar Glycerolipid Classes in Marine Plankton Using Triple Quadrupole MS. Lipids 48:185-195. Doi:10.1007/s11745-012-3748-0



Last updated: April 3, 2015
 


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