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MB Sample ID: SA327634
| Local Sample ID: | Sp03 |
| Subject ID: | SU003131 |
| Subject Type: | Mammal |
| Subject Species: | Florida manatee (Trichechus manatus latirostris) |
| Age Or Age Range: | Adult |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003131 |
| Subject Type: | Mammal |
| Subject Species: | Florida manatee (Trichechus manatus latirostris) |
| Age Or Age Range: | Adult |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Sp03 | SA327634 | FL038871 | Crystal River | Capture Location |
| Sp03 | SA327634 | FL038871 | NP | Reproductive Status |
| Sp03 | SA327634 | FL038871 | F | Sex |
Collection:
| Collection ID: | CO003124 |
| Collection Summary: | Blood samples were collected from wild FL manatees at manatee health assessments. Blood was collected in 7-mL lithium heparinized vacutainers and centrifuged at 3200 rpm for 10 min onsite at atmospheric temperature. The plasma supernatant was transferred to cryovials in 1 mL aliquots. Plasma samples were kept on dry ice during transport and stored at -80 °C at USGS and University of Florida (UF) until analysis. |
| Collection Protocol Filename: | Collection Summary.txt |
| Sample Type: | Blood (plasma) |
| Storage Conditions: | -80℃ |
| Collection Vials: | lithium heparinized vacutainers |
| Storage Vials: | Cryovials |
Treatment:
| Treatment ID: | TR003140 |
| Treatment Summary: | Plasma was not treated. Lipids were measured and compared between pregnant and non-pregnant manatees. |
Sample Preparation:
| Sampleprep ID: | SP003137 |
| Sampleprep Summary: | Lithium heparinized manatee plasma samples, quality control samples, extraction and solvent blanks, and pooled group samples were extracted via the Folch extraction method (2:1 chloroform: methanol) (Folch et al., 1957). The plasma samples were thawed on ice and briefly homogenized. Next, 50 µL of each sample was added to a glass test tube. Three quality control samples were prepared the same manner using 50 µL of SRM 1950. All samples, except the solvent blanks, were spiked with 50 µL of deuterium-labeled internal standards from 10 different lipid classes at known concentrations (diluted 1:10) (Splash Lipidomix, Avanti Polar Lipids, Alabaster, AL). Biphasic extraction was conducted by adding 3 mL of 2:1 chloroform:methanol solution to each test tube including the quality control samples and extraction blanks. The samples were then vortexed for 10 s, 500 µL of Optima-grade water was added, and the tubes were inverted 5 times. All samples were centrifuged at 2500 g for 5 min at 4 °C. The resulting bottom lipid containing layer was then removed and transferred to a new glass test tube using glass Pasteur pipettes. Samples were divided into sub-groups determined by study site, sex, and reproductive status. Extract pools were created by equal contributions from each sample within a group. Finally, the samples, controls, and pools were evaporated under a steady stream of nitrogen at 25 °C for about 25 min. The samples were then reconstituted with 100 µL of isopropanol and vortexed for 10 s. The reconstituted solutions were then transferred to autosampler vial inserts inside glass autosampler vials for UHPLC-MS/MS analysis. |
| Sampleprep Protocol Filename: | Sample Prep.txt |
Combined analysis:
| Analysis ID | AN004950 | AN004951 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters ACQUITY UPLC BEH C18(100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH C18(100 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak area | Peak area |
Chromatography:
| Chromatography ID: | CH003735 |
| Chromatography Summary: | The extracted lipid samples, pooled groups, quality controls, and blanks were analyzed by UHPLC-MS/MS on a Thermo Vanquish UHPLC system coupled to a Thermo Q-Exactive Orbitrap mass spectrometer. The column used was a Waters Acquity BEH, 100 mm x 2.1 mm x 1.7 µm. Mobile phase A and B contained 60:40 acetonitrile/water (v/v) and 90:10 isopropanol/acetonitrile (v/v) respectively, with both phases containing 5mM ammonium formate and 0.1% formic acid. The mobile phase gradient was as follows: a linear increase of solvent B from 40% to 55% over 7 min, then increased to 65% by min 8, held at 65% for 4 min, linear increase to 95% over 8 min, increased to 100% over 2 min, solvent B was held at 100% for 5 min before decreasing to 40% in a tenth of a min. Finally, solvent B was held at 100% for another 3 min. The sample injection volume was 10 µL at a flow rate of 0.25 mL/min, the column compartment was 45 °C, and the autosampler temperature was 10 °C. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC BEH C18(100 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | a linear increase of solvent B from 40% to 55% over 7 min, then increased to 65% by min 8, held at 65% for 4 min, linear increase to 95% over 8 min, increased to 100% over 2 min, solvent B was held at 100% for 5 min before decreasing to 40% in a tenth of a min. Finally, solvent B was held at 100% for another 3 min. |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 60:40 acetonitrile/water (v/v), 5mM ammonium formate and 0.1% formic acid |
| Solvent B: | 90:10 isopropanol/acetonitrile (v/v), 5mM ammonium formate and 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004690 |
| Analysis ID: | AN004950 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | All samples were analyzed in full scan positive and negative ion modes with spray voltages +3.0kV and -3.0kV, respectively, at 70,000 resolution at m/z 200, the scan range was m/z 100-1500 and source temperature at 300 °C. Tandem mass spectra were collected via criterion based on data-dependent acquisition for the top-10 ions with stepped normalized collision energy 20, 25, and 30, an isolation window of 1Da, dynamic exclusion of 6 s, and using IE-Omics for the generation of exclusion lists in iterative-exclusion experiments (Koelmel, Kroeger, Gill, et al., 2017). Extracted Lipid Data Analysis LipidMatch software was implemented to identify and integrate the detected lipids (Koelmel, Kroeger, Ulmer, et al., 2017). The peak areas were normalized relative to the internal standards of the same subclass as the lipid of interest. For lipids that did not have an internal standard of the same subclass, the internal standard with the closest chemical structure was used (i.e., internal standard with the same head group). Otherwise, the internal standard with the closest chromatographic retention time (same ionization polarity) was used for analytes without a corresponding internal standard of the same lipid subclass. The plasma lipid concentrations were reported as µg/mL. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | MS Metadata.txt |
| MS ID: | MS004691 |
| Analysis ID: | AN004951 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | All samples were analyzed in full scan positive and negative ion modes with spray voltages +3.0kV and -3.0kV, respectively, at 70,000 resolution at m/z 200, the scan range was m/z 100-1500 and source temperature at 300 °C. Tandem mass spectra were collected via criterion based on data-dependent acquisition for the top-10 ions with stepped normalized collision energy 20, 25, and 30, an isolation window of 1Da, dynamic exclusion of 6 s, and using IE-Omics for the generation of exclusion lists in iterative-exclusion experiments (Koelmel, Kroeger, Gill, et al., 2017). Extracted Lipid Data Analysis LipidMatch software was implemented to identify and integrate the detected lipids (Koelmel, Kroeger, Ulmer, et al., 2017). The peak areas were normalized relative to the internal standards of the same subclass as the lipid of interest. For lipids that did not have an internal standard of the same subclass, the internal standard with the closest chemical structure was used (i.e., internal standard with the same head group). Otherwise, the internal standard with the closest chromatographic retention time (same ionization polarity) was used for analytes without a corresponding internal standard of the same lipid subclass. The plasma lipid concentrations were reported as µg/mL. |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | MS Metadata.txt |
