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MB Sample ID: SA045298
| Local Sample ID: | Tgon_HKKO_13Cglucose_t120_A |
| Subject ID: | SU000843 |
| Subject Type: | Cells (protozoan parasite) |
| Subject Species: | Toxoplasma gondii |
| Taxonomy ID: | 5811 |
| Genotype Strain: | RH (Type I strain) |
| Cell Biosource Or Supplier: | ATCC |
| Subject Comments: | Asexual stage (Tachyzoite) |
| Cell Passage Number: | In continuous culture |
| Cell Counts: | 10^8 cells per sample |
| Species Group: | Microorganism |
Select appropriate tab below to view additional metadata details:
Combined analysis:
| Analysis ID | AN001295 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Accela |
| Column | Synergy Hydro-RP (Phenomenex) and Accucore C18 (Thermo) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE |
| Units | NA |
Chromatography:
| Chromatography ID: | CH000907 |
| Chromatography Summary: | The acetonitrile:water extracts from parasites were dried under nitrogen flow and resuspended in 200 µls of water:methanol (97:3) containing 10 mM tributylamine and 15 mM acetic acid. This solvent was also used as buffer A and methanol as buffer B for liquid chromatography using a Synergy Hydro-RP column (Phenomenex) with a bed volume of 100 mm x 2 mm and particle size of 2.5 µ. For some samples, an alternate method, using a Thermo Accucore C18 column with a bed volume of 150 mm x 2.1 mm and 2.6 µ particle size. A solvent system composed of water buffered with 0.1 % formic acid (buffer A) and acetonitrile (buffer B), was used on a 20 minute gradient run with a flow rate of 200 µl/min as follows- hold at 10% acetonitrile for 30 seconds and gradually ramp up to 15%, 20%, 50%, 60% and 90% acetonitrile by 3, 6, 10, 12, 13 minutes, hold at 90% acetonitrile till 15 minutes, ramp down to 10% acetonitrile by 15.5 minutes and hold till 20 minutes. LC-MS analysis was done using a Exactive Orbitrap mass spectrometer, coupled to an Accela U-HPLC (Thermo Fisher Scientific) and HTC PAL autosampler (CTC Analytics AG). The mass spectrometer was run in negative mode, scanning a mass-charge ratio (m/z) range of 85-1000. All other parameters used for LC-MS instrumentation in this study were similar to published protocols. |
| Instrument Name: | Thermo Accela |
| Column Name: | Synergy Hydro-RP (Phenomenex) and Accucore C18 (Thermo) |
| Column Pressure: | 250 to 400 bar |
| Column Temperature: | 22 deg. Cel. |
| Flow Gradient: | A solvent system composed of water buffered with 0.1 % formic acid (buffer A) and acetonitrile (buffer B), was used on a 20 minute gradient run with a flow rate of 200 µl/min as follows- hold at 10% acetonitrile for 30 seconds and gradually ramp up to 15%, 20%, 50%, 60% and 90% acetonitrile by 3, 6, 10, 12, 13 minutes, hold at 90% acetonitrile till 15 minutes, ramp down to 10% acetonitrile by 15.5 minutes and hold till 20 minutes. |
| Flow Rate: | 200 µl/min |
| Injection Temperature: | 22 deg. Cel. |
| Internal Standard: | PIPES @ 100ng/ml final concentration |
| Sample Injection: | 10 µl |
| Solvent A: | 97% water/3% methanol; 0.1% formic acid; 15 mM acetic acid; 10 mM tributylamine |
| Solvent B: | Methanol for Synergy Hydro-RP column. Acetonitril for accucore C18 column. |
| Analytical Time: | 20 minutes |
| Preconditioning: | 5-10 minutes |
| Time Program: | 20 minutes |
| Transferline Temperature: | 22 deg. Cel. |
| Washing Buffer: | Methanol |
| Sample Loop Size: | 10 µl |
| Sample Syringe Size: | 100 µl |
| Chromatography Type: | Reversed phase |
