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.

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Study IDST001304
Study TitleMulti-omics analysis delineates the distinct functions of sub-cellular acetyl-CoA pools in Toxoplasma gondii
Study SummaryAcetyl-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 NameSiddiqui
First NameGhizal
Address381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia
Emailghizal.siddiqui@monash.edu
Phone99039282
Submit Date2020-01-16
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2020-03-03
Release Version1
Ghizal Siddiqui Ghizal Siddiqui
https://dx.doi.org/10.21228/M8938R
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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
SA094349DDACS_ACLko_1_shieldNo shield
SA094350DDACS_3_shieldNo shield
SA094351Ku80_3_shieldNo shield
SA094352Ku80_1_shieldNo shield
SA094353DDACS_2_shieldNo shield
SA094354DDACS_1_shieldNo shield
SA094355Ku80_2_shieldNo shield
SA094356DDACS_ACLko_2_shieldNo shield
SA094357DDACS_ACLko_3_shieldNo shield
SA094358Ku80_3plusshieldplus shield
SA094359Ku80_2plusshieldplus shield
SA094360Ku80_1_plusshieldplus shield
SA094361DDACS_1plusshieldplus shield
SA094362DDACS_ACLko_1plusshieldplus shield
SA094363DDACS_ACLko_2plusshieldplus shield
SA094364DDACS_ACLko_3plusshieldplus shield
SA094365DDACS_2plusshieldplus shield
SA094366DDACS_3plusshieldplus 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
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