Summary of Study ST001304
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 PR000885. The data can be accessed directly via it's Project DOI: 10.21228/M8938R This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001304 |
| Study Title | Multi-omics analysis delineates the distinct functions of sub-cellular acetyl-CoA pools in Toxoplasma gondii |
| Study Summary | Acetyl-CoA is a key metabolite in all organisms, implicated in transcriptional regulation, post-translational modification as well as fuelling the TCA-cycle and the synthesis and elongation of fatty acids (FAs). The obligate intracellular parasite Toxoplasma gondii possesses two enzymes which produce acetyl-CoA in the cytosol and nucleus: acetyl-CoA synthetase (ACS) and ATP-citrate lyase (ACL), while the branched-chain α-keto acid dehydrogenase-complex (BCKDH) generates acetyl-CoA in the mitochondrion. To obtain a global and integrative picture of the role of distinct sub-cellular acetyl-CoA pools, we measured the acetylome, transcriptome, proteome and metabolome of parasites lacking ACL/ACS or BCKDH. Loss of ACL/ACS results in the hypo-acetylation of nucleo-cytosolic and secretory proteins, alters gene expression broadly and is required for the synthesis of parasite-specific FAs. In contrast, loss of BCKDH causes few specific changes in the acetylome, transcriptome and proteome which allow these parasites to rewire their metabolism to adapt to the obstruction of the TCA-cycle. |
| Institute | Monash University |
| Last Name | Siddiqui |
| First Name | Ghizal |
| Address | 381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia |
| ghizal.siddiqui@monash.edu | |
| Phone | 99039282 |
| Submit Date | 2020-01-16 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2020-03-03 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR000885 |
| Project DOI: | doi: 10.21228/M8938R |
| Project Title: | Multi-omics analysis delineates the distinct functions of sub-cellular acetyl-CoA pools in Toxoplasma gondii |
| Project Summary: | Acetyl-CoA is a key metabolite in all organisms, implicated in transcriptional regulation, post-translational modification as well as fuelling the TCA-cycle and the synthesis and elongation of fatty acids (FAs). The obligate intracellular parasite Toxoplasma gondii possesses two enzymes which produce acetyl-CoA in the cytosol and nucleus: acetyl-CoA synthetase (ACS) and ATP-citrate lyase (ACL), while the branched-chain α-keto acid dehydrogenase-complex (BCKDH) generates acetyl-CoA in the mitochondrion. To obtain a global and integrative picture of the role of distinct sub-cellular acetyl-CoA pools, we measured the acetylome, transcriptome, proteome and metabolome of parasites lacking ACL/ACS or BCKDH. Loss of ACL/ACS results in the hypo-acetylation of nucleo-cytosolic and secretory proteins, alters gene expression broadly and is required for the synthesis of parasite-specific FAs. In contrast, loss of BCKDH causes few specific changes in the acetylome, transcriptome and proteome which allow these parasites to rewire their metabolism to adapt to the obstruction of the TCA-cycle. |
| Institute: | Monash University |
| Last Name: | Siddiqui |
| First Name: | Ghizal |
| Address: | 381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia |
| Email: | ghizal.siddiqui@monash.edu |
| Phone: | 99039282 |
Subject:
| Subject ID: | SU001378 |
| Subject Type: | Cultured cells |
| Subject Species: | Toxoplasma gondii |
| Taxonomy ID: | 5811 |
Factors:
Subject type: Cultured cells; Subject species: Toxoplasma gondii (Factor headings shown in green)
| mb_sample_id | local_sample_id | treatment |
|---|---|---|
| SA094349 | DDACS_ACLko_1_shield | No shield |
| SA094350 | DDACS_3_shield | No shield |
| SA094351 | Ku80_3_shield | No shield |
| SA094352 | Ku80_1_shield | No shield |
| SA094353 | DDACS_2_shield | No shield |
| SA094354 | DDACS_1_shield | No shield |
| SA094355 | Ku80_2_shield | No shield |
| SA094356 | DDACS_ACLko_2_shield | No shield |
| SA094357 | DDACS_ACLko_3_shield | No shield |
| SA094358 | Ku80_3plusshield | plus shield |
| SA094359 | Ku80_2plusshield | plus shield |
| SA094360 | Ku80_1_plusshield | plus shield |
| SA094361 | DDACS_1plusshield | plus shield |
| SA094362 | DDACS_ACLko_1plusshield | plus shield |
| SA094363 | DDACS_ACLko_2plusshield | plus shield |
| SA094364 | DDACS_ACLko_3plusshield | plus shield |
| SA094365 | DDACS_2plusshield | plus shield |
| SA094366 | DDACS_3plusshield | plus shield |
| Showing results 1 to 18 of 18 |
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 |
