Summary of study ST001204

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 PR000809. The data can be accessed directly via it's Project DOI: 10.21228/M83X38 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001204
Study TitlePeroxide antimalarial extended treatment timecourse on trophozoite-stage P. falciparum parasites
Study SummaryRed blood cells (RBCs) infected with trophozoite stage P. falciparum parasites (3D7 strain) at 10% parasitaemia and 2% haematocrit were treated with OZ277 (300 nM), OZ439 (300 nM), DHA (100 nM) or vehicle (0.03% DMSO). This was a 4-timepoint study, with samples taken 0, 3, 6 and 9 h after drug or vehicle addition. Samples treated with vehicle acted as the untreated control. Samples from drug treated uninfected RBCs were also taken to ensure the observed drug effects were parasite specific.
Institute
Monash University
Last NameGiannangelo
First NameCarlo
Address381 Royal Parade, Parkville, Victoria, 3052, Australia
Emailcarlo.giannangelo@monash.edu
Phone99039282
Submit Date2019-06-25
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2019-07-17
Release Version1
Carlo Giannangelo Carlo Giannangelo
https://dx.doi.org/10.21228/M83X38
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000809
Project DOI:doi: 10.21228/M83X38
Project Title:System-wide biochemical analysis reveals ozonide and artemisinin antimalarials initially act by disrupting malaria parasite haemoglobin digestion
Project Summary:Artemisinins are currently the first-line antimalarials, and rely on a peroxide pharmacophore for their potent activity. OZ277 (arterolane) and OZ439 (artefenomel) are newer synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a “multi-omics” workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of peroxide-treated P. falciparum infected red blood cells (iRBCs) revealed a rapid depletion of short Hb-derived peptides, while untargeted peptidomics showed accumulation of longer Hb peptides. Quantitative proteomics and ABPP assays demonstrated that Hb digesting proteases were significantly increased in abundance and activity following treatment, respectively. The association between peroxide activity and Hb catabolism was also confirmed in a K13-mutant artemisinin resistant parasite line. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in peroxide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short peroxide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate peroxide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage peroxide-induced damage.
Institute:Monash University
Last Name:Giannangelo
First Name:Carlo
Address:381 Royal Parade, Parkville, Victoria, 3052, Australia
Email:carlo.giannangelo@monash.edu
Phone:99039282

Subject:

Subject ID:SU001271
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum;Homo sapiens
Taxonomy ID:5833/9606
Genotype Strain:3D7
Age Or Age Range:28-34 h post invasion

Factors:

Subject type: Cultured cells; Subject species: Plasmodium falciparum;Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id cell_type treatment treatment_duration_(h)
SA084646DHA_iRBC_0h_BiRBC DHA -
SA084647DHA_iRBC_0h_CiRBC DHA -
SA084648DHA_iRBC_0h_DiRBC DHA -
SA084649DHA_iRBC_3h_CiRBC DHA 3
SA084650DHA_iRBC_3h_BiRBC DHA 3
SA084651DHA_iRBC_3h_DiRBC DHA 3
SA084652DHA_iRBC_6h_BiRBC DHA 6
SA084653DHA_iRBC_6h_DiRBC DHA 6
SA084654DHA_iRBC_6h_CiRBC DHA 6
SA084655DHA_iRBC_9h_DiRBC DHA 9
SA084656DHA_iRBC_9h_BiRBC DHA 9
SA084657DHA_iRBC_9h_CiRBC DHA 9
SA084658DMSO_iRBC_0h_BiRBC DMSO -
SA084659DMSO_iRBC_0h_DiRBC DMSO -
SA084660DMSO_iRBC_0h_CiRBC DMSO -
SA084661DMSO_iRBC_3h_CiRBC DMSO 3
SA084662DMSO_iRBC_3h_DiRBC DMSO 3
SA084663DMSO_iRBC_3h_BiRBC DMSO 3
SA084664DMSO_iRBC_6h_CiRBC DMSO 6
SA084665DMSO_iRBC_6h_DiRBC DMSO 6
SA084666DMSO_iRBC_6h_BiRBC DMSO 6
SA084667DMSO_iRBC_9h_DiRBC DMSO 9
SA084668DMSO_iRBC_9h_BiRBC DMSO 9
SA084669DMSO_iRBC_9h_CiRBC DMSO 9
SA084670OZ277_iRBC_0h_BiRBC OZ277 -
SA084671OZ277_iRBC_0h_DiRBC OZ277 -
SA084672OZ277_iRBC_0h_CiRBC OZ277 -
SA084673OZ277_iRBC_3h_CiRBC OZ277 3
SA084674OZ277_iRBC_3h_BiRBC OZ277 3
SA084675OZ277_iRBC_3h_DiRBC OZ277 3
SA084676OZ277_iRBC_6h_CiRBC OZ277 6
SA084677OZ277_iRBC_6h_BiRBC OZ277 6
SA084678OZ277_iRBC_6h_DiRBC OZ277 6
SA084679OZ277_iRBC_9h_BiRBC OZ277 9
SA084680OZ277_iRBC_9h_CiRBC OZ277 9
SA084681OZ277_iRBC_9h_DiRBC OZ277 9
SA084682OZ439_iRBC_0h_CiRBC OZ439 -
SA084683OZ439_iRBC_0h_DiRBC OZ439 -
SA084684OZ439_iRBC_0h_BiRBC OZ439 -
SA084685OZ439_iRBC_3h_DiRBC OZ439 3
SA084686OZ439_iRBC_3h_BiRBC OZ439 3
SA084687OZ439_iRBC_3h_CiRBC OZ439 3
SA084688OZ439_iRBC_6h_DiRBC OZ439 6
SA084689OZ439_iRBC_6h_CiRBC OZ439 6
SA084690OZ439_iRBC_6h_BiRBC OZ439 6
SA084691OZ439_iRBC_9h_DiRBC OZ439 9
SA084692OZ439_iRBC_9h_CiRBC OZ439 9
SA084693OZ439_iRBC_9h_BiRBC OZ439 9
SA084694DHA_unRBC_0h_DunRBC DHA -
SA084695DHA_unRBC_0h_CunRBC DHA -
SA084696DHA_unRBC_0h_BunRBC DHA -
SA084697DHA_unRBC_3h_CunRBC DHA 3
SA084698DHA_unRBC_3h_BunRBC DHA 3
SA084699DHA_unRBC_3h_DunRBC DHA 3
SA084700DHA_unRBC_6h_DunRBC DHA 6
SA084701DHA_unRBC_6h_BunRBC DHA 6
SA084702DHA_unRBC_6h_CunRBC DHA 6
SA084703DHA_unRBC_9h_CunRBC DHA 9
SA084704DHA_unRBC_9h_DunRBC DHA 9
SA084705DHA_unRBC_9h_BunRBC DHA 9
SA084706DMSO_unRBC_0h_CunRBC DMSO -
SA084707DMSO_unRBC_0h_DunRBC DMSO -
SA084708DMSO_unRBC_0h_BunRBC DMSO -
SA084709DMSO_unRBC_3h_BunRBC DMSO 3
SA084710DMSO_unRBC_3h_DunRBC DMSO 3
SA084711DMSO_unRBC_3h_CunRBC DMSO 3
SA084712DMSO_unRBC_6h_BunRBC DMSO 6
SA084713DMSO_unRBC_6h_DunRBC DMSO 6
SA084714DMSO_unRBC_6h_CunRBC DMSO 6
SA084715DMSO_unRBC_9h_BunRBC DMSO 9
SA084716DMSO_unRBC_9h_CunRBC DMSO 9
SA084717DMSO_unRBC_9h_DunRBC DMSO 9
SA084718OZ277_unRBC_0h_CunRBC OZ277 -
SA084719OZ277_unRBC_0h_DunRBC OZ277 -
SA084720OZ277_unRBC_0h_BunRBC OZ277 -
SA084721OZ277_unRBC_3h_BunRBC OZ277 3
SA084722OZ277_unRBC_3h_DunRBC OZ277 3
SA084723OZ277_unRBC_3h_CunRBC OZ277 3
SA084724OZ277_unRBC_6h_DunRBC OZ277 6
SA084725OZ277_unRBC_6h_BunRBC OZ277 6
SA084726OZ277_unRBC_6h_CunRBC OZ277 6
SA084727OZ277_unRBC_9h_BunRBC OZ277 9
SA084728OZ277_unRBC_9h_DunRBC OZ277 9
SA084729OZ277_unRBC_9h_CunRBC OZ277 9
SA084730OZ439_unRBC_0h_DunRBC OZ439 -
SA084731OZ439_unRBC_0h_BunRBC OZ439 -
SA084732OZ439_unRBC_0h_CunRBC OZ439 -
SA084733OZ439_unRBC_3h_DunRBC OZ439 3
SA084734OZ439_unRBC_3h_CunRBC OZ439 3
SA084735OZ439_unRBC_3h_BunRBC OZ439 3
SA084736OZ439_unRBC_6h_CunRBC OZ439 6
SA084737OZ439_unRBC_6h_DunRBC OZ439 6
SA084738OZ439_unRBC_6h_BunRBC OZ439 6
SA084739OZ439_unRBC_9h_CunRBC OZ439 9
SA084740OZ439_unRBC_9h_BunRBC OZ439 9
SA084741OZ439_unRBC_9h_DunRBC OZ439 9
Showing results 1 to 96 of 96

Collection:

Collection ID:CO001265
Collection Summary:Infected RBCs were adjusted to 10% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 1E8 cells were pelleted by centrifugation at 1,000 x g for 3 min and the culture medium was removed. Parasite metabolism was quenched by the addition of ice-cold PBS, pelleted again and the supernatant discarded prior to metabolite extraction. Metabolites were extracted from the cell pellet using 150 µL of cold methanol. The extraction solvent containing the internal standard compounds CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid) and TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) was directly added to the cell pellet, mixed by pipetting and subjected to automatic vortex mixing for 1 h at 4°C. Following the 1 h incubation, samples were pelleted by centrifugation at 21,100 x g for 10 min, 110 µL of particle free supernatant was transferred to glass LC-MS vials and stored at -80°C until analysis. A 15 µL aliquot of each sample was combined to generate a pooled biological quality control (PBQC) sample.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR001286
Treatment Summary:Trophozoite-stage P. falciparum infected RBCs (10% parasitaemia and 2% Hct) were treated with OZ277 (300 nM), OZ439 (300 nM), DHA (100 nM) or an equivalent volume of DMSO (0.03%) for 0, 3, 6 and 9 h. During the drug incubation period parasites were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2.
Treatment Compound:OZ277 (arterolane), OZ439 (artefenomel) and dihydroartemisinin (DHA)
Treatment Vehicle:DMSO
Cell Media:Complete RPMI medium (10.4 g/L) containing HEPES (5.94 g/L), hypoxanthine (50 mg/L), sodium bicarbonate (2.1 g/L) and Albumax II (5 g/L).
Cell Media Lastchanged:Immediately prior to initiation of drug incubation

Sample Preparation:

Sampleprep ID:SP001279
Sampleprep Summary:Infected RBCs were adjusted to 10% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 1E8 cells were pelleted by centrifugation at 1,000 x g for 3 min and the culture medium was removed. Parasite metabolism was quenched by the addition of ice-cold PBS, pelleted again and the supernatant discarded prior to metabolite extraction. Metabolites were extracted from the cell pellet using 150 µL of cold methanol. The extraction solvent containing the internal standard compounds CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid) and TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) was directly added to the cell pellet, mixed by pipetting and subjected to automatic vortex mixing for 1 h at 4°C. Following the 1 h incubation, samples were pelleted by centrifugation at 21,100 x g for 10 min, 110 µL of particle free supernatant was transferred to glass LC-MS vials and stored at -80°C until analysis. A 15 µL aliquot of each sample was combined to generate a pooled biological quality control (PBQC) sample.
Processing Storage Conditions:Described in summary
Extract Storage:Described in summary

Combined analysis:

Analysis ID AN002004 AN002005
Analysis type MS MS
Chromatography type pHILIC pHILIC
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)
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 Peak intensity Peak intensity

Chromatography:

Chromatography ID:CH001450
Chromatography Summary:The 32 min gradient HPLC run was from 80% B to 50% B over 15 min, then to 5% B at 18 min, followed by a wash with 5% B for 3 min and re-equilibrated with 80% B at a flow rate of 0.3 mL/min.
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)
Solvent A:20 mM ammonium carbonate
Solvent B:100% acetonitrile
Chromatography Type:pHILIC

MS:

MS ID:MS001857
Analysis ID:AN002004
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times.
Ion Mode:POSITIVE
  
MS ID:MS001858
Analysis ID:AN002005
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times.
Ion Mode:NEGATIVE
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