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MB Sample ID: SA071424
| Local Sample ID: | 8379 |
| 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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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 |
