Summary of Study ST002181

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 PR001388. The data can be accessed directly via it's Project DOI: 10.21228/M89H7H 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.

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Study IDST002181
Study TitlePiperaquine-resistant PfCRT mutations differentially impact drug transport, hemoglobin catabolism and parasite physiology in Plasmodium falciparum asexual blood stages.
Study SummaryThe emergence of multidrug resistance in Plasmodium falciparum parasites presents a significant obstacle to the malaria elimination agenda. Resistance to piperaquine (PPQ), an important first-line partner drug, has spread across Southeast Asia where it has contributed to widespread treatment failures. The genetic cause of resistance to PPQ is attributable to a novel set of amino acid substitutions in the P. falciparum chloroquine resistance transporter (PfCRT). In this study, we used magnetically-purified trophozoite extracts from seven different lines comprising five genetically-modified and two field isolates. Three independent extractions with triplicate technical repeats were run by mass spectrometry on positive and negative modes and spectral peak raw data analyzed to obtain the fold change difference between the samples and parental control, Dd2Dd2crt. We show that PPQ-resistant, PfCRT mutant asexual blood stage parasites accumulate higher levels of hemoglobin-derived peptides than do their PPQ-sensitive counterparts.
Institute
Pennsylvania State University
DepartmentDepartment of Biochemistry and Molecular Biology
Last NameLlinas
First NameManuel
AddressW126 Millenium Science Complex, University Park, PA 16802
Emailmanuel@psu.edu
Phone814-867-3527
Submit Date2022-05-18
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2022-10-19
Release Version1
Manuel Llinas Manuel Llinas
https://dx.doi.org/10.21228/M89H7H
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001388
Project DOI:doi: 10.21228/M89H7H
Project Title:Piperaquine-resistant PfCRT mutations differentially impact drug transport, hemoglobin catabolism and parasite physiology in Plasmodium falciparum asexual blood stages.
Project Summary:The emergence of multidrug resistance in Plasmodium falciparum parasites presents a significant obstacle to the malaria elimination agenda. Resistance to piperaquine (PPQ), an important first-line partner drug, has spread across Southeast Asia where it has contributed to widespread treatment failures. The genetic cause of resistance to PPQ is attributable to a novel set of amino acid substitutions in the P. falciparum chloroquine resistance transporter (PfCRT). In this study, we used magnetically-purified trophozoite extracts from seven different lines comprising five genetically-modified and two field isolates. Three independent extractions with triplicate technical repeats were run by mass spectrometry on positive and negative modes and spectral peak raw data analyzed to obtain the fold change difference between the samples and parental control, Dd2Dd2crt. We show that PPQ-resistant, PfCRT mutant asexual blood stage parasites accumulate higher levels of hemoglobin-derived peptides than do their PPQ-sensitive counterparts.
Institute:The Pennsylvania State University
Department:Department of Biochemistry and Molecular Biology
Last Name:Llinas
First Name:Manuel
Address:W126 Millenium Science Complex, University Park, PA 16802
Email:manuel@psu.edu
Phone:814-867-3527

Subject:

Subject ID:SU002267
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum
Taxonomy ID:5833
Genotype Strain:DD2B2
Species Group:Unicellular parasites

Factors:

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

mb_sample_id local_sample_id PfCRT Haplotype
SA209611Dd2GC03crt-10242018-23D7
SA209612Dd2GC03crt-10242018-13D7
SA209613Dd2GC03crt-10292018-13D7
SA209614Dd2GC03crt-10312018-23D7
SA209615Dd2GC03crt-10242018-33D7
SA209616Dd2GC03crt-10312018-13D7
SA209617Dd2GC03crt-10312018-33D7
SA209618Dd2GC03crt-10292018-33D7
SA209619Dd2GC03crt-10292018-23D7
SA209620Dd2Dd2crt-10272018-2Dd2
SA209621Dd2B2-11082018-1Dd2
SA209622Dd2Dd2crt-10272018-1Dd2
SA209623Dd2Dd2crt-10272018-3Dd2
SA209624Dd2Dd2crt-10062018-2Dd2
SA209625Dd2B2-11102018-2Dd2
SA209626Dd2B2-11102018-3Dd2
SA209627Dd2B2-11102018-1Dd2
SA209628Dd2B2-11082018-3Dd2
SA209629Dd2B2-11082018-2Dd2
SA209630Dd2B2-11122018-1Dd2
SA209631Dd2B2-11122018-2Dd2
SA209632Dd2Dd2crt-10012018-3Dd2
SA209633Dd2Dd2crt-10062018-1Dd2
SA209634Dd2Dd2crt-10012018-2Dd2
SA209635Dd2Dd2crt-10012018-1Dd2
SA209636Dd2B2-11122018-3Dd2
SA209637Dd2Dd2crt-10062018-3Dd2
SA209638Dd2Dd2crtF145I-06282018-1Dd2+F145I
SA209639Dd2Dd2crtF145I-06302018-1Dd2+F145I
SA209640Dd2Dd2crtF145I-06282018-3Dd2+F145I
SA209641Dd2Dd2crtF145I-06282018-2Dd2+F145I
SA209642Dd2Dd2crtF145I-06302018-2Dd2+F145I
SA209643Dd2Dd2crtF145I-06302018-3Dd2+F145I
SA209644Dd2Dd2crtF145I-07042018-2Dd2+F145I
SA209645Dd2Dd2crtF145I-07042018-1Dd2+F145I
SA209646Dd2Dd2crtF145I-07042018-3Dd2+F145I
SA209647Dd2Dd2crtG353V-07282018-2Dd2+G353V
SA209648Dd2Dd2crtG353V-08022018-1Dd2+G353V
SA209649Dd2Dd2crtG353V-08022018-2Dd2+G353V
SA209650Dd2Dd2crtG353V-07282018-3Dd2+G353V
SA209651Dd2Dd2crtG353V-07212018-3Dd2+G353V
SA209652Dd2Dd2crtG353V-07212018-1Dd2+G353V
SA209653Dd2Dd2crtG353V-07212018-2Dd2+G353V
SA209654Dd2Dd2crtG353V-08022018-3Dd2+G353V
SA209655Dd2Dd2crtG353V-07282018-1Dd2+G353V
SA209656RF12-11152018-2Dd2+H97Y
SA209657RF12-11152018-1Dd2+H97Y
SA209658RF12-11132018-3Dd2+H97Y
SA209659RF12-11152018-3Dd2+H97Y
SA209660RF12-11172018-1Dd2+H97Y
SA209661RF12-11172018-3Dd2+H97Y
SA209662RF12-11172018-2Dd2+H97Y
SA209663RF12-11132018-2Dd2+H97Y
SA209664RF12-11132018-1Dd2+H97Y
SA209665RF7-07232018-2Dd2+M343L
SA209666RF7-07312018-2Dd2+M343L
SA209667RF7-07312018-1Dd2+M343L
SA209668RF7-09102018-3Dd2+M343L
SA209669RF7-07312018-3Dd2+M343L
SA209670RF7-09102018-2Dd2+M343L
SA209671RF7-09102018-1Dd2+M343L
SA209672RF7-07232018-3Dd2+M343L
SA209673Dd2Dd2crtM343L-10092018-1Dd2+M343L
SA209674Dd2Dd2crtM343L-10132018-1Dd2+M343L
SA209675Dd2Dd2crtM343L-10092018-3Dd2+M343L
SA209676Dd2Dd2crtM343L-10092018-2Dd2+M343L
SA209677Dd2Dd2crtM343L-10132018-2Dd2+M343L
SA209678Dd2Dd2crtM343L-10132018-3Dd2+M343L
SA209679Dd2Dd2crtM343L-10152018-3Dd2+M343L
SA209680Dd2Dd2crtM343L-10152018-2Dd2+M343L
SA209681Dd2Dd2crtM343L-10152018-1Dd2+M343L
SA209682RF7-07232018-1Dd2+M343L
Showing results 1 to 72 of 72

Collection:

Collection ID:CO002260
Collection Summary:Mycoplasma-free parasites were sorbitol-synchronized in each generation for at least two generations followed by magnetic enrichment of 32 hr post-invasion trophozoites using MACS CS columns on a SuperMACS™ II Separator (Miltenyi Biotec, Inc.) to remove uninfected RBCs. Trophozoite counts were determined on a hemocytometer. Parasites were lysed in 1 mL of 90% cold methanol, containing 0.5 μM of the internal standard [13C4, 15N1]-Aspartate (Cambridge Isotope) to correct for technical variations arising from sample processing. Samples were vortexed to disrupt cell pellets and to generate uniform homogenates, which were centrifuged (13,000´ g for 10 min) and the supernatants then harvested. Supernatants were dried under nitrogen prior to resuspension in HPLC-grade water for LC-MS analysis.
Sample Type:Plasmodium cells

Treatment:

Treatment ID:TR002279
Treatment Summary:Cells were all untreated, but had different genetic backgrounds.

Sample Preparation:

Sampleprep ID:SP002273
Sampleprep Summary:Supernatants were dried under nitrogen prior to resuspension in HPLC-grade water for LC-MS analysis.
Processing Method:Lysis
Processing Storage Conditions:4℃
Extraction Method:See SA Cobbold et al JBC 2013
Extract Cleanup:Clarification via centrifugation followed by nitrogen drying
Extract Storage:-80℃
Sample Resuspension:HPLC-grade water spiked with chlorpropamide
Sample Spiking:Internal Standard 13C4, 15N1-Aspartate used for sample preparation normalizer

Combined analysis:

Analysis ID AN003572 AN003573
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Prominence 20 UFLCXR Prominence 20 UFLCXR
Column Waters Acquity BEH C18 (100 x 2mm,1.7um) Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type ESI ESI
MS instrument type Triple TOF Triple TOF
MS instrument name ABI Sciex 5600 TripleTOF ABI Sciex 5600 TripleTOF
Ion Mode NEGATIVE POSITIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH002641
Chromatography Summary:Samples (5ul) were separated by reverse phase HPLC using a Prominence 20 UFLCXR system (Shimadzu, Columbia, MD) with a Waters (Milford, MA) BEH C18 column (100mm x 2.1mm 1.7 um particle size) maintained at 55C and a 20 minute aqueous acetonitrile gradient, at a flow rate of 250 ul/min. Solvent A was HPLC grade water with 0.1% formic acid and Solvent B was HPLC grade acetonitrile with 0.1% formic acid. The initial condition were 97% A and 3 % B, increasing to 45% B at 10 min, 75% B at 12 min where it was held at 75% B until 17.5 min before returning to the initial conditions.
Instrument Name:Prominence 20 UFLCXR
Column Name:Waters Acquity BEH C18 (100 x 2mm,1.7um)
Column Temperature:55
Flow Gradient:The initial conditions were 97% A and 3 % B, increasing to 45% B at 10 min, 75% B at 12 min where it was held at 75% B until 17.5 min before returning to the initial conditions.
Flow Rate:250 ul/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS003329
Analysis ID:AN003572
Instrument Name:ABI Sciex 5600 TripleTOF
Instrument Type:Triple TOF
MS Type:ESI
MS Comments:The eluate was delivered into a 5600 (QTOF) TripleTOF using a Duospray™ ion source (all Sciex, Framingham, MA).The capillary voltage was set at 5.5 kV in positive/negative ion mode with a declustering potential of 80V. The mass spectrometer was operated with a 100 ms TOF scan from 50 to 1000 m/z, and 16 MS/MS product ion scans (100 ms) per duty cycle using a collision energy of 50V with a 20V spread.
Ion Mode:NEGATIVE
  
MS ID:MS003330
Analysis ID:AN003573
Instrument Name:ABI Sciex 5600 TripleTOF
Instrument Type:Triple TOF
MS Type:ESI
MS Comments:The eluate was delivered into a 5600 (QTOF) TripleTOF using a Duospray™ ion source (all Sciex, Framingham, MA).The capillary voltage was set at 5.5 kV in positive/negative ion mode with a declustering potential of 80V. The mass spectrometer was operated with a 100 ms TOF scan from 50 to 1000 m/z, and 16 MS/MS product ion scans (100 ms) per duty cycle using a collision energy of 50V with a 20V spread.
Ion Mode:POSITIVE
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