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MB Sample ID: SA094352
| Local Sample ID: | Ku80_1_shield |
| Subject ID: | SU001378 |
| Subject Type: | Cultured cells |
| Subject Species: | Toxoplasma gondii |
| Taxonomy ID: | 5811 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001378 |
| Subject Type: | Cultured cells |
| Subject Species: | Toxoplasma gondii |
| Taxonomy ID: | 5811 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Ku80_1_shield | SA094352 | FL013482 | No shield | treatment |
Collection:
| Collection ID: | CO001373 |
| Collection Summary: | Freshly egressing parasites were extracted through repeated syringe lysis (3x, 28G), purified from host cell material through filtration (3 μm pore size, Millipore/Merck) and pelleted by centrifugation (2,800g, 20 min, 4 °C). Pellets were washed with ice-cold PBS (3x) and metabolites were extracted in 250 μl chloroform:methanol:water (1:3:1). Samples were vortexed vigorously for 30 min at 4 °C and the insoluble material was pelleted through centrifugation (21,000g, 6 min, 4 °C). Samples were transferred to glass high-performance liquid chromatography (HPLC) vials and stored at −80 °C until analysis. |
| Collection Protocol Comments: | Sample Source/Type details: Human Foreskin Fibroblasts |
| Sample Type: | Fibroblasts |
Treatment:
| Treatment ID: | TR001393 |
| Treatment Summary: | Intracellular parasites were incubated in the either the absence or presence of Shld-1 for 16 hours before samples collected |
Sample Preparation:
| Sampleprep ID: | SP001386 |
| Sampleprep Summary: | Pellets were washed with ice-cold PBS (3x) and metabolites were extracted in 250 μl chloroform:methanol:water (1:3:1). Samples were vortexed vigorously for 30 min at 4 °C and the insoluble material was pelleted through centrifugation (21,000g, 6 min, 4 °C). Samples were transferred to glass high-performance liquid chromatography (HPLC) vials and stored at −80 °C until analysis. |
Combined analysis:
| Analysis ID | AN002172 | AN002173 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RSLC | Thermo Dionex Ultimate 3000 RSLC |
| Column | ZIC-pHILIC,Merck | ZIC-pHILIC,Merck |
| 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 | Signal Intensity | Signal Intensity |
Chromatography:
| Chromatography ID: | CH001590 |
| Instrument Name: | Thermo Dionex Ultimate 3000 RSLC |
| Column Name: | ZIC-pHILIC,Merck |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002021 |
| Analysis ID: | AN002172 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | DPSOHV ȝ/ ZHUH injected onto a Dionex RSLC U3000 LC system (Thermo) fitted with a ZIC-pHILIC column ȝP SDUWLFOH VL]H E\ PP 0HUFN DQG P0 DPPRQLXP FDUERQDWH $ DQG acetonitrile (B) were used as the mobile phases. A 30 min gradient starting from 80% B to 40% B over 20 min, followed by washing at 5% B for 3 min and re-equilibration at 80% B, was used. MS utilised a Q-Exactive Orbitrap MS (Thermo) with a heated electrospray source operating in positive and negative modes (rapid switching) and a mass resolution of 35,000 from m/z 85 to 1,050. Sample injections within the experiment were randomized to avoid any impact of systematic instrument drift on metabolite signals. Retention times for ~350 authentic standards were checked manually to aid metabolite identification. Metabolomics data sets were analysed using IDEOM. Raw files were converted to mzXML with msconvert, extraction of LC-MS peak signals was conducted with the Centwave algorithm in XCMS, alignment of samples and filtering of artefacts with mzMatch, and additional data filtering and metabolite identification in IDEOM. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002022 |
| Analysis ID: | AN002173 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | DPSOHV ȝ/ ZHUH injected onto a Dionex RSLC U3000 LC system (Thermo) fitted with a ZIC-pHILIC column ȝP SDUWLFOH VL]H E\ PP 0HUFN DQG P0 DPPRQLXP FDUERQDWH $ DQG acetonitrile (B) were used as the mobile phases. A 30 min gradient starting from 80% B to 40% B over 20 min, followed by washing at 5% B for 3 min and re-equilibration at 80% B, was used. MS utilised a Q-Exactive Orbitrap MS (Thermo) with a heated electrospray source operating in positive and negative modes (rapid switching) and a mass resolution of 35,000 from m/z 85 to 1,050. Sample injections within the experiment were randomized to avoid any impact of systematic instrument drift on metabolite signals. Retention times for ~350 authentic standards were checked manually to aid metabolite identification. Metabolomics data sets were analysed using IDEOM. Raw files were converted to mzXML with msconvert, extraction of LC-MS peak signals was conducted with the Centwave algorithm in XCMS, alignment of samples and filtering of artefacts with mzMatch, and additional data filtering and metabolite identification in IDEOM. |
| Ion Mode: | NEGATIVE |
