Summary of Study ST000817
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000582. The data can be accessed directly via it's Project DOI: 10.21228/M8FD4K This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST000817 |
| Study Title | Dynamics of metabolism, proliferation and differentiation in Toxoplasma gondii mutants deficient in glycolysis and gluconeogenesis |
| Study Type | Time course 13C labeling of Toxoplasma gondii using glucose and glutamine |
| Study Summary | The focus of this study was to profile the metabolic fates of glucose and glutamine in wild type, glycolytic mutant (hexokinase KO) and gluconeogenesis mutant (PEPCK1 KO) tachyzoite stage T. gondii parasites. 13C6-glucose and 13C5-15N1-glutamine were used as metabolic tracers. Metabolites from glycolysis, pentosephosphate pathway and Kerbs cycle were identified and their isotopomer abundance was quantified in order to visualize metabolic flux across the indicated pathways. A time course (from 0 minutes to 120 minutes) analysis was carried out in the labeling experiments. Here, host cell free extracellular tachyzoite stage T. gondii parasites were used. |
| Institute | CSIR-National Chemical Laboratory |
| Department | Biochemical Sciences Division |
| Laboratory | Molecular Parasitology Laboratory |
| Last Name | Shanmugam |
| First Name | Dhanasekaran |
| Address | Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India |
| d.shanmugam@ncl.res.in | |
| Phone | +91-20-25902719 |
| Submit Date | 2017-07-22 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2017-10-11 |
| Release Version | 1 |
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 |
