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MB Sample ID: SA071459

Local Sample ID:8381
Subject ID:SU001094
Subject Type:Fish
Subject Species:Oreochromis mossambicus
Taxonomy ID:8127
Age Or Age Range:1 to 14 years
Weight Or Weight Range:0.1 to 5.6 kg
Height Or Height Range:NA
Gender:Male and female

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Subject:

Subject ID:SU001094
Subject Type:Fish
Subject Species:Oreochromis mossambicus
Taxonomy ID:8127
Age Or Age Range:1 to 14 years
Weight Or Weight Range:0.1 to 5.6 kg
Height Or Height Range:NA
Gender:Male and female

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
8381SA071459FL011207MHGroup
8381SA071459FL011207MGender

Collection:

Collection ID:CO001088
Collection Summary:Sample Collection and Metadata Fifty one (n=51) Mozambique tilapia (Oreochromis mossambicus) were captured at various locations along the inflow of Loskop Dam, Mpumalanga, South Africa (25°28'22.8 S: 29°15'25.2 E) between May 2-6, 2016. On capture, blood was immediately drawn and collected in lithium heparin 3 mL vacutainers (BD vacutainer, Franklin Lakes, NJ). After the first blood draw, fish were kept in aerated tanks and transported back to the field station for necropsy. All fish work was conducted using the permit and animal handling protocol that was reviewed and approved by the Mpumalanga Tourism and Parks Agency (Project #ES 6/1 ). Upon arrival to the field station, blood was immediately centrifuged in collection tubes and 500 μL of plasma was aliquoted into cryogenic storage vials (Corning, Corning, NY) for lipidomic studies (n=51). Adipose tissue was removed during dissection and flash frozen and stored for lipidomics analysis. Juvenile fish with an ambiguous sex assignment were excluded. Pictures of the whole fish and individual biopsied tissues were taken under consistent lighting in a photo box for documentation. A subset of the sampled fish, representing individuals with varying degrees of disease severity with matched age, size, and weight, were selected for lipidomics of adipose tissue (n=31).
Sample Type:Blood (plasma)
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001108
Treatment Summary:Mozambique tilapia were not treated, but rather captured in the wild.

Sample Preparation:

Sampleprep ID:SP001101
Sampleprep Summary:Sample Preparation for Liquid Chromatography - Mass Spectrometric Analysis Lipid internal standards were purchased from the following locations to generate a lipid internal standard mix: Nu-Chek Prep (CE 19:0, DG 19:2/19:2, DG 20:0/20:0, MG 17:0, TG 13:0/13:0/13:0, TG 17:1/17:1/17:1, and TG 19:0/19:0/19:0), and Avanti Polar Lipids (Cer d18:1/25:0, free cholesterol-d7, GalCer d18:1/12:0, GlcCer d18:1/12:0, LPA 17:0, LPC 19:0, LPE 14:0, LSM d17:1, OxPC 16:0/9:0, PA 14:0/14:0, PC 14:1/14:1, PC 19:0/19:0, PE 15:0/15:0, PE 17:0/17:0, PG 15:0/15:0, PG 17:0/17:0, PI 8:0/8:0, PS 14:0/14:0, and SM d18:1/6:0). Optima methanol and HPLC grade chloroform were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HPLC grade 2-propanol was acquired from Alfa Aesar (Haverhill, MA, USA). Ultrapure water with 18 MΩ cm resistivity (Millipore Milli-Q Gradient A10; EMD Millipore, Billerica, MA, USA) was used for sample preparation. Ammonium acetate and analytical grade formic acid were purchased from Fisher Scientific. All mobile phase solvents were Fisher Optima LC/MS grade (acetonitrile, isopropanol, and water). Plasma samples (50 μL) were extracted using the Bligh–Dyer extraction protocol.1 Briefly, samples were removed from the −80 °C freezer and thawed on ice. To each sample, a 4 mL chloroform:methanol mixture (1:1, v:v) was added followed by a 1.75 mL water addition, accounting for the water present in plasma. Samples were then incubated on ice for 30 min, vortexed for 20 s, and centrifuged at 2000 rpm for 10 min to separate the organic and aqueous layers. The organic layer was collected and the aqueous layer was re-extracted with 2 mL 1:1 (v:v, chloroform:methanol). The organic layers were combined, evaporated under nitrogen, and reconstituted in 50 μL of isopropanol for a 5 μL injection. The final average concentration of internal standards in mg standard per kg plasma wet weights were: CE(19:0) (1.06 mg/kg), Cer(d18:1/25:0) (116.99 mg/kg), DG(19:2/19:2) (0.08 mg/kg), DG(20:0/20:0) (0.73 mg/kg), GlcCer(d18:1/12:0) (0.13 mg/kg), LPA(17:0) (0.35 mg/kg), LPC(19:0) (2.48 mg/kg), LPE(14:0) (1.49 mg/kg), PA(14:0/14:0) (8.81 mg/kg), PC(14:1/14:1) (2.72 mg/kg), PC(19:0/19:0) (3.53 mg/kg), PE(15:0/15:0) (1.16 mg/kg), PE(17:0/17:0) (3.09 mg/kg), PG(15:0/15:0) (1.25 mg/kg), PG(17:0/17:0) (2.52 mg/kg), PS(14:0/14:0) (1.84 mg/kg), SM(d18:1/6:0) (0.15 mg/kg), TG(17:1/17:1/17:1) (1.53 mg/kg), and TG(19:0/19:0/19:0) (1.22 mg/kg). Whole adipose tissues were cryopulverized using a Retsch Cryomill (Retsch, Haan, Germany) and 30 mg aliquots of powder were removed for extraction. All powdered aliquots were extracted using the Folch method (2:1, v/v chloroform:methanol).2 Briefly, 100 μL of the internal standard mix was spiked into tissue samples on ice. Chloroform (2 mL) and methanol (1 mL) were added to all samples. Samples were vortexed and incubated on ice for 30 minutes. Upon the addition of water (750 μL) to induce phase separation, samples were vortexed and incubated on ice for an additional 10 min. Samples were centrifuged for 10 min at 2000 rpm. The organic layer was removed and the aqueous layer was re-extracted with 1500 µL of chloroform:methanol (2:1, v/v). The organic layers were combined, evaporated under nitrogen, and reconstituted in 2000 µL of isopropanol for a 2 μL injection. The final average concentrations of internal standards in adipose tissues were: CE(19:0) (71.40 mg/kg), Cer(d18:1/25:0) (7875.37 mg/kg), DG(19:2/19:2) (5.71 mg/kg), DG(20:0/20:0) (48.81 mg/kg), GalCer(d18:1/12:0) (8.86 mg/kg), GlcCer(d18:1/12:0) (8.87 mg/kg), LPA(17:0) (23.32 mg/kg), LPC(19:0) (167.27 mg/kg), LPE(14:0) (100.23 mg/kg), MG(17:0) (15.92 mg/kg), PA(14:0/14:0) (593.08 mg/kg), PC(14:1/14:1) (183.25 mg/kg), PC(19:0/19:0) (237.29 mg/kg), PE(15:0/15:0) (78.05 mg/kg), PE(17:0/17:0) (208.09 mg/kg), PG(15:0/15:0) (84.15 mg/kg), PG(17:0/17:0) (169.34 mg/kg), PI(8:0/8:0) (1.21 mg/kg), PS(14:0/14:0) (123.92 mg/kg), SM(d18:1/6:0) (9.91 mg/kg), TG(13:0/13:0/13:0) (86.90 mg/kg), TG(17:1/17:1/17:1) (103.22 mg/kg), and TG(19:0/19:0/19:0) (82.01 mg/kg). Data Acquisition All lipid extracts were randomized and analyzed by ultra-high performance liquid chromatography coupled to a high-resolution mass spectrometer (UHPLC-HRMS). Mass spectra were acquired on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with a heated electrospray ionization (HESI II) probe in positive and negative ion mode. HESI and mass spectrometric parameters for lipid extracts were as follows in positive/negative ion mode, respectively: spray voltage: 3.5/2.5 kV, sheath gas: 40/35 arbitrary units, auxiliary nitrogen pressure: 15 arbitrary units, sweep gas: 1/0 arbitrary units, ion transfer tube and vaporizer temperatures: 325 and 300/275 °C, and RF lens level: 30. Full scan, data-dependent MS/MS (ddMS2-top10), and data-independent acquisition mode (specifically all-ion fragmentation (AIF)) data were collected at m/z 150–2000, corresponding to the mass range of most expected cellular lipids. For ddMS2-top10, iterative exclusion,3 where lipids selected for fragmentation are excluded from fragmentation in a sequential injection, was applied. This method drastically improves lipid coverage.3 External calibration was applied before each run to allow for full scan LC-HRMS analysis at 120,000 resolution (m/z 200 FWHM). A Thermo Scientific Vanquish UHPLC system (Thermo Scientific, San Jose, CA) was coupled to the Orbitrap Fusion Lumos Tribrid for the chromatographic separation of lipids. The autosampler temperature was maintained at 4 °C for all experiments. Solvent extraction blanks and quality control samples were jointly analyzed over the course of a batch (10–15 samples). A Waters Acquity C18 BEH column (2.1 × 100 mm, 1.7 μm particle size, Waters, Milford, MA) maintained at 60 °C was used for all lipidomic studies. The mobile phase flow rate was 450 μL/min. The gradient program consisted of mobile phase C [60:40 acetonitrile/water] and mobile phase D [90:8:2 isopropanol/ acetonitrile/water], each containing 10 mM ammonium formate and 0.1% formic acid. The gradient included 32% D at 0 min, 40% D at 1 min, a hold at 40% D until 1.5 min, 45% D at 4 min, 50% D at 5 min, 60% D at 8 min, 70% D at 11 min, 80% D at 14 min, 100% D at 16 min, and a hold at 100% D until 17 min. The total run time was 22 min, which included a 5-min equilibration. (1) Bligh, E. G.; Dyer, W. J. A Rapid Method of Total Lipid Extraction and Purification. Can. J. Biochem. Physiol. 1959, 37 (8), 911–917. (2) Folch, J.; Lees, M.; Sloane Stanley, G. H. A Simple Method for the Isolation and Purification of Total Lipides from Animal Tissues. J. Biol. Chem. 1957, 226 (1), 497–509. (3) Koelmel, J. P.; Kroeger, N. M.; Gill, E. L.; Ulmer, C. Z.; Bowden, J. A.; Patterson, R. E.; Yost, R. A.; Garrett, T. J. Expanding Lipidome Coverage Using LC-MS/MS Data-Dependent Acquisition with Automated Exclusion List Generation. J. Am. Soc. Mass Spectrom. 2017, 28 (5), 908–917.
Processing Storage Conditions:-80℃
Extraction Method:Folch

Combined analysis:

Analysis ID AN001721
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Vanquish
Column Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Fusion Tribrid Orbitrap
Ion Mode UNSPECIFIED
Units mg/kg (relative quantification, NOT ABSOLUTE)

Chromatography:

Chromatography ID:CH001217
Chromatography Summary:A Thermo Scientific Vanquish UHPLC system (Thermo Scientific, San Jose, CA) was coupled to the Orbitrap Fusion Lumos Tribrid for the chromatographic separation of lipids. The autosampler temperature was maintained at 4 °C for all experiments. Solvent extraction blanks and quality control samples were jointly analyzed over the course of a batch (10–15 samples). A Waters Acquity C18 BEH column (2.1 × 100 mm, 1.7 μm particle size, Waters, Milford, MA) maintained at 60 °C was used for all lipidomic studies. The mobile phase flow rate was 450 μL/min. The gradient program consisted of mobile phase C [60:40 acetonitrile/water] and mobile phase D [90:8:2 isopropanol/ acetonitrile/water], each containing 10 mM ammonium formate and 0.1% formic acid. The gradient included 32% D at 0 min, 40% D at 1 min, a hold at 40% D until 1.5 min, 45% D at 4 min, 50% D at 5 min, 60% D at 8 min, 70% D at 11 min, 80% D at 14 min, 100% D at 16 min, and a hold at 100% D until 17 min. The total run time was 22 min, which included a 5-min equilibration.
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
Column Temperature:60 °C
Flow Gradient:The gradient included 32% D at 0 min, 40% D at 1 min, a hold at 40% D until 1.5 min, 45% D at 4 min, 50% D at 5 min, 60% D at 8 min, 70% D at 11 min, 80% D at 14 min, 100% D at 16 min, and a hold at 100% D until 17 min. The total run time was 22 min, which included a 5-min equilibration.
Flow Rate:450 uL/min
Solvent A:40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Solvent B:90% isopropanol/8% acetonitrile/2% water; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:Reversed phase

MS:

MS ID:MS001593
Analysis ID:AN001721
Instrument Name:Thermo Fusion Tribrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:UNSPECIFIED
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