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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:
Subject:
| 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 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Tgon_HKKO_13Cglucose_t120_A | SA045298 | FL009055 | T. gondii RH HKKO | Cell line |
Collection:
| Collection ID: | CO000837 |
| Collection Summary: | Extracellular tachyzoite stage Toxoplasma gondii parasites incubated in either +13C6-glucose or +13C5-15N2-glutamine containing DMEM medium for 0, 5, 10, 30, 60 & 120 minutes were collected by centrifugation and immediately extracted with chilled acetonitrile:water (80:20) mixture and further process for LC-MS analysis. |
| Sample Type: | Cells |
| Collection Method: | Centrifuging cell suspension at 5000 rpm for 5 min at 4 deg. Cel. |
| Collection Time: | At 0, 5, 10, 30, 60 & 120 minutes post labeling with either +13C6-glucose or +13C5-15N2-glutamine. |
| Volumeoramount Collected: | 10^8 cells per sample |
| Storage Conditions: | -80 deg. Cel. Freezer post extraction until LC-MS analysis can be carried out. |
| Collection Vials: | 1.5 ml capped tubes |
| Storage Vials: | 1.5 ml capped tubes |
| Collection Tube Temp: | 4 deg. Cel. |
Treatment:
| Treatment ID: | TR000857 |
| Treatment Summary: | Tachyzoite stage Toxoplasma gondii parasites were freshly isolated from infected Human Foreskin Fibroblast monolayers and the extracellular parasites were incubated in DMEM medium supplemented with either +13C6-glucose or +13C5-15N2-glutamine. Treatment with labeled substrates was done for 0, 5, 10, 30, 60 & 120 minutes before harvesting and processing the cells for LC-MS analysis. |
| Cell Storage: | Liquid nitrogen cell storage system |
| Cell Growth Container: | Standard incubator maintaining 37 deg. Cel. & 5% CO2 |
| Cell Media: | DMEM minus glucose medium (cat. No. 11966; Gibco, ThermoFisher Scientific) supplemented with +13C6-glucose. DMEM minus glutamine medium (cat. No. 11960; Gibco, ThermoFisher Scientific) supplemented with +13C5-15N2-glutamine. |
Sample Preparation:
| Sampleprep ID: | SP000850 |
| Sampleprep Summary: | Metabolic labeling, metabolite extraction, and LC-MS profiling – DMEM minus glucose media supplemented with +613C glucose (5.5 mM) and MEM minus glutamine media supplemented with +513C-+213N glutamine (4 mM) were used as culture mediums for metabolic labeling studies using RH wt, RH ∆ku80, RH ∆hk, and RH ∆pepck1 parasites. Freshly isolated extracellular (host cell free) tachyzoites stage parasites were washed once in complete medium and resuspended in either 13C labeled glucose or glutamine (Cambridge Isotope Laboratories, Inc.) containing medium at a density of 108 parasites per milliliter. Labeling was allowed to proceed over a time period of 5, 10, 15, 30, 60 and 120 minutes in 1 ml culture suspension. At each time point, metabolites were extracted from replicate parasite samples using a modified version of a previously reported protocol. Briefly, host cell free parasites were collected by centrifugation at 3000 rpm for 5 minutes in cold and then immediately resuspended in 200 µls of ice cold 80% acetonitrile (Chem-Impex; JT Bakers) in water and incubated in ice for 15 minutes with intermittent vortexing. The supernatant was then collected after centrifuging at 13,000 rpm for 5 minutes and the pellet was further extracted twice with 100 µls of the same solvent using ultrasound in a sonicating iced water bath for 15 minutes. All the extracts were pooled (total 400 µls) and stored in -80°C until further processing for liquid chromatography coupled mass spectrometry (LC-MS) analysis. |
| Extraction Method: | Hydrophilic organic extract |
| Extract Storage: | -80 deg. Cel. until LC-MS analysis is done |
| Sample Resuspension: | 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. |
| Sample Derivatization: | n/a |
| Sample Spiking: | PIPES @ 100ng/ml final concentration |
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 |
MS:
| MS ID: | MS001188 |
| Analysis ID: | AN001295 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 320 deg. Cel |
| Capillary Voltage: | -3.0KV |
| Ion Source Temperature: | 250 DEG. Cel. |
| Ionization: | Negative |
| Mass Accuracy: | <1ppm |
| Reagent Gas: | Nitrogen |
| Dataformat: | .Raw |
| Resolution Setting: | 70000 |
| Scan Range Moverz: | 100 to 1000 Da |
| Scanning Range: | Full Ms |
