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MB Sample ID: SA185594
| Local Sample ID: | p0.5_PA |
| Subject ID: | SU002063 |
| Subject Type: | Yeast |
| Subject Species: | Candida albicans |
| Taxonomy ID: | 5476 |
| Genotype Strain: | SC 5314 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002063 |
| Subject Type: | Yeast |
| Subject Species: | Candida albicans |
| Taxonomy ID: | 5476 |
| Genotype Strain: | SC 5314 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| p0.5_PA | SA185594 | FL022943 | AbA-treated | Treatment |
| p0.5_PA | SA185594 | FL022943 | 0.5 | Concentration (ug/ml) |
| p0.5_PA | SA185594 | FL022943 | Phospholipids | Extraction method |
| p0.5_PA | SA185594 | FL022943 | Positive | Ionization mode |
Collection:
| Collection ID: | CO002056 |
| Collection Summary: | A starter culture was prepared by inoculating two loopfuls of C. albicans yeast colony in 5 ml of yeast peptone dextrose (YPD) broth and incubated at 37°C for 24 hours. 150 microlitres of the starter culture was then inoculated into 150 ml fresh YPD broth (10^7 of cells/ml) and let to grow for 6 hours until it reached 10^9 of cells/ml. |
| Sample Type: | Yeast cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002075 |
| Treatment Summary: | The yeast cells were exposed to different concentration of Aureobasidin A (0.5, 1, 2 and 4 microgram/ml). DMSO-treated yeast culture was used as a control. The cultures were incubated for another 3 hours prior to harvesting. Each of the conditions was performed in duplicate. |
Sample Preparation:
| Sampleprep ID: | SP002069 |
| Sampleprep Summary: | Lipids enrichment Lipids were enriched using a method as described by Guan and Wenk (2010). After AbA treatment, the yeast cells were harvested and washed twice. The wet weight was standardized. Briefly, the cells were resuspended in 2 ml 95% ethanol: water: diethyl ether: pyridine: ammonium hydroxide (15 : 15 : 5 : 1 : 0.018). The cells were broken by glass beads (vortexed twice for 1 minute each) and incubated for 20 min at 60 °C. Debris was pelleted by centrifugation and the supernatant was transferred to a fresh tube. The pellet was re-extracted once more using the same procedure. The pooled supernatants were divided into equal aliquots and dried using CentriVap Concentrator System at 50 °C. One aliquot was used for phospholipids and the other for sphingolipids analysis. Phospholipids extraction For phospholipid extraction, the dried lipid film was desalted by butanol extraction using 300 µl of water-soluble butanol and 150 µl of sterile distilled water. The mixture was vortexed and centrifuged. The top layer was pooled and dried in CentriVap Concentrator System at 4°C. The dried phospholipids were resuspended in 400 µl chloroform and methanol (1:1, v/v), vortexed for 30 s and were centrifuged again at 10,000 rpm for 5 min before injecting into liquid chromatography system. Sphingolipids extraction A fraction enriched in sphingolipids was obtained by mild alkaline hydrolysis, which degrades ester linkages found in many glycerophospholipids (Brockerhoff, 1963). To achieve this, the dried lipid films were resuspended in 400 µl chloroform: methanol: water (16 : 16 : 5, v/v/v). Glycerophospholipids were deacylated by 400 µl of 0.2 N NaOH and incubated at 30 °C for 45 minutes. 400 µl 0.5 M EDTA was added and the samples were neutralized with 80 µl of 1 N acetic acid. 400 µl of chloroform was added before the samples were vortexed and centrifuged. Sphingolipids were pooled by collecting the lower phase of the layers and it was dried using CentriVap Concentrator System at 4°C. The lipid extract was then desalted using butanol extraction as described above. Liquid Chromatography-Mass spectrometry (LC-MS) The LC-MS of the C. albicans lipids were performed using a 1260 Infinity High Performance Liquid Chromatography system coupled with a 6540 UHD Accurate-Mass Q-TOF mass spectrometer from Agilent Technologies with a Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI) source. Typically, 2 µl of sample was injected for mass spectrometry analysis. The Dual AJS ESI capillary voltage and nozzle voltage was maintained at 3.0 kV and 1 kV, respectively. The gas temperature was maintained at 300 °C, drying gas flow was set at the rate of 8 L/min, sheath gas temperature and sheath gas flow at 350 °C and 11 L/min respectively and nebulizer pressure was set at 35 psi. The mass spectrum was acquired from a mass-to charge ratio (m/z) of 400–1400 in the positive and negative ion mode, with an acquisition time of 3 minutes, and the scan duration was 1 second. Samples were directly infused using an autosampler syringe pump at a flow rate of 10 µl/min into Zorbax Eclipse Plus C18, 2.1 x 100 mm, 1.8 µm reverse phase column. The mobile phase was chloroform and methanol with 1 : 1 (v/v ratio) and water with 0.1% formic acid at a flow rate of 15 µl/min. Individual molecular species was identified using tandem mass spectrometry and in general, the collision energy used was in the range 25–80 eV. Two reference masses were used in each ionization modes, i.e., 121.0509 m/z and 922.0098 m/z for positive ionization, 112.9855 m/z and 1033.9881 m/z for negative ionization mode. All the data attained from mass spectral was in a d. format. |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | Described in summary |
Combined analysis:
| Analysis ID | AN003232 | AN003233 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Agilent 6530 | Agilent 6530 |
| Column | Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) | Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Agilent 6540 QTOF | Agilent 6540 QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | minute | minute |
Chromatography:
| Chromatography ID: | CH002384 |
| Instrument Name: | Agilent 6530 |
| Column Name: | Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) |
| Column Temperature: | 45 |
| Flow Gradient: | yes |
| Flow Rate: | 15ul/ml |
| Internal Standard: | 121.0509 m/z and 922.0098 m/z for positive ionization, 112.9855 m/z and 1033.9881 m/z for negative ionization mode. |
| Solvent A: | Chloroform; methanol |
| Solvent B: | Water; formic acids |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003006 |
| Analysis ID: | AN003232 |
| Instrument Name: | Agilent 6540 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Agilent MassHunter Workstation Qualitative Analysis software version B.06.00 |
| Ion Mode: | POSITIVE |
| MS ID: | MS003007 |
| Analysis ID: | AN003233 |
| Instrument Name: | Agilent 6540 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Agilent MassHunter Workstation Qualitative Analysis software version B.06.00 |
| Ion Mode: | NEGATIVE |
