Summary of Study ST004222

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 PR002663. The data can be accessed directly via it's Project DOI: 10.21228/M8F545 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.

Study ID	ST004222
Study Title	Elucidating the source of elevated proline in APR parasites
Study Summary	In this study, we use a custom Triplex labeled media, containing stable isotope labeled 15-N proline (Pro+1), 13C,15N-arginine (Arg+2), and 13C5-glutamine (Gln+5) to identify the source of elevated proline levels in APR parasites. Labeled proline levels were assessed in Dd2 (WT) parasites and Dd2-APR parasites (parasites previously selected with HFG that demonstrate increased intracellular proline or the APR). High levels of labeled proline (+1) implicate import from the extracellular environment as a primary driver of the APR; high levels of labeled proline (+2) implicate the arginine to proline biosynthetic pathway as the primary source; and high levels of labeled proline (+5) implicate the glutamine to proline biosynthetic pathway as the primary source. Unlabeled proline (+0) indicates proline derived from hemoglobin digestion (either directly as proline, or via the arginine and/or glutamine conversion pathways).
Institute	Broad Institute of MIT and Harvard
Department	Metabolomics Platform
Last Name	Clish
First Name	Clary
Address	300 Binney Street, Cambridge, MA 02142
Email	clary@broadinstitute.org
Phone	617-714-7654
Submit Date	2025-09-08
Num Groups	2
Total Subjects	7
Study Comments	Study 1 of 5
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-10-07
Release Version	1

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Project:

Project ID:	PR002663
Project DOI:	doi: 10.21228/M8F545
Project Title:	Loss of P. falciparum Amino Acid Transporter (ApiAT2) Function Increases Intracellular Proline and Confers Resistance to Prolyl-tRNA Synthetase Inhibitors
Project Summary:	Plasmodium falciparum evades the antimalarial activity of proline-competitive prolyl-tRNA synthetase (PfProRS) inhibitors, such as halofuginone (HFG), by a unique resistance mechanism termed the Adaptive Proline Response (APR). The APR is characterized by a marked elevation of intracellular proline following drug exposure. Contrary to initial expectations, the APR is not mediated by alterations in canonical proline metabolic pathways involving arginase (PfARG) and ornithine aminotransferase (PfOAT). Instead, we identified loss-of-function mutations in the Apicomplexan Amino acid Transporter 2 (PfApiAT2) as the primary genetic driver of this resistance phenotype. Importantly, reversion of these mutations to wildtype effectively suppresses the APR, establishing PfApiAT2 as the molecular determinant of this novel resistance mechanism. The elucidation of the APR significantly advances our understanding of antimalarial drug resistance. By delineating the role of PfApiAT2 in this process, we establish critical insights for the development of strategies to circumvent PfProRS inhibitor resistance for future antimalarial therapies.
Institute:	Broad Institute of MIT and Harvard
Department:	Metabolomics Platform
Last Name:	Clish
First Name:	Clary
Address:	300 Binney Street, Cambridge, MA, 02142, USA
Email:	clary@broadinstitute.org
Phone:	617-714-7654
Funding Source:	NIH-NIAID R01AI143723 and R21AI132981; NIH-NIGMS T32 GM008666 and F31AI129412; Bill and Melinda Gates Foundation OPP1132451 and OPP1086203; and the Harvard Defeating Malaria Initiative
Project Comments:	5 experiements
Contributors:	Selina Bopp, Lọla Fagbami, Amy Deik, Claudia Taccheri, Akansha Pant, Madeline Luth, Daisy Chen, Mark A. Tye, Imran Ullah, Robert Morris, Wilhelm Haas, Elizabeth A. Winzeler, Clary B. Clish, Amanda K. Lukens, Ralph Mazitschek, Dyann F. Wirth

Subject:

Subject ID:	SU004374
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833
Species Group:	Unicellular parasites

Factors:

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

mb_sample_id	local_sample_id	Sample source	Parasite_type	Parasite_line	Timepoint
SA485905	Study1_Dd2APR_iRBC_10h_R1	infected RBC (iRBC)	Dd2 IND	Dd2APR	10 h
SA485906	Study1_Dd2APR_iRBC_10h_R2	infected RBC (iRBC)	Dd2 IND	Dd2APR	10 h
SA485907	Study1_Dd2APR_iRBC_10h_R3	infected RBC (iRBC)	Dd2 IND	Dd2APR	10 h
SA485908	Study1_Dd2APR_iRBC_10h_R4	infected RBC (iRBC)	Dd2 IND	Dd2APR	10 h
SA485909	Study1_Dd2_iRBC_10h_R1	infected RBC (iRBC)	Dd2 WT	Dd2	10 h
SA485910	Study1_Dd2_iRBC_10h_R2	infected RBC (iRBC)	Dd2 WT	Dd2	10 h
SA485911	Study1_Dd2_iRBC_10h_R3	infected RBC (iRBC)	Dd2 WT	Dd2	10 h

Showing results 1 to 7 of 7

Showing results 1 to 7 of 7

Collection:

Collection ID:	CO004367
Collection Summary:	Highly synchronous (within 4 h) late trophozoite stage parasites were magnetically purified with MACS LD columns (Miltenyi Biotec Inc., San Diego, CA, USA). Each sample was incubated for 10hr with either labeled or unlabeled media, harvested by centrifugation, washed twice in PBS, and then suspended in 10 μL PBS (Life Technologies, Carlsbad, CA, USA). Polar metabolites were extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (0.2 ng/μL valine-d8 (Sigma Aldrich, St. Louis, MO, USA); and 0.2 ng/μL phenylalanine-d8 (Cambridge Isotope Laboratories, Tewksbury, MA, USA) and stored at -80°C prior to the metabolite profiling assays.
Sample Type:	Cultured cells
Collection Location:	Harvard Chan School of Public Health, 665 Huntington Ave. Boston, MA 02115
Volumeoramount Collected:	100 uL
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR004383
Treatment Summary:	Cultures were cultured in 5% human O+ hematocrit and maintained under standard conditions [RPMI 1640 (Life Technologies) supplemented with 28 mM NaHCO3 (Sigma), 25 mM HEPES (Sigma), 50 mg/mL hypoxanthine (Sigma), 25 μg/mL gentamycin (Sigma), and 0.5% AlbuMAX II (Life Technologies)] until preparation for treatment. In preparation for treatment, 25 mL synchronized parasites culture at ~5% parasitemia and 32-36hr staging at beginning of experiment were pelleted and resuspended in 4mL serum-free RPMI (Incomplete Media). Parasites were magnetically purified with an LD column (Miltenyi Biotec Inc., San Diego, CA, USA) and washed until the flow thru ran clear. The column was removed from the magnet and each sample eluted in 2mL incomplete RPMI. Samples were divided into two microfuge tubes, each receiving half (1 mL) of the cell suspension. Cells were pelleted and resuspended in either incomplete RPMI (unlabeled) or our Triplex labeled RPMI media (RPMI media first prepared without proline, glutamine, or arginine and then supplemented with isotope labeled amino acids at regular RPMI concentrations: 15N-proline (Pro+1; 0.17 mM), 13C,15N-arginine (Arg+2; 0.38 mM), and 13C5-gluatmine (Gln+5; 2.05 mM)). Parasites were incubated in labeled or unlabeled media for 10h at 37ºC under standard malaria gas mixture. Samples were harvested by centrifugation, washed twice, and then polar metabolites extracted.

Treatment ID:

TR004383

Treatment Summary:

Cultures were cultured in 5% human O+ hematocrit and maintained under standard conditions [RPMI 1640 (Life Technologies) supplemented with 28 mM NaHCO3 (Sigma), 25 mM HEPES (Sigma), 50 mg/mL hypoxanthine (Sigma), 25 μg/mL gentamycin (Sigma), and 0.5% AlbuMAX II (Life Technologies)] until preparation for treatment. In preparation for treatment, 25 mL synchronized parasites culture at ~5% parasitemia and 32-36hr staging at beginning of experiment were pelleted and resuspended in 4mL serum-free RPMI (Incomplete Media). Parasites were magnetically purified with an LD column (Miltenyi Biotec Inc., San Diego, CA, USA) and washed until the flow thru ran clear. The column was removed from the magnet and each sample eluted in 2mL incomplete RPMI. Samples were divided into two microfuge tubes, each receiving half (1 mL) of the cell suspension. Cells were pelleted and resuspended in either incomplete RPMI (unlabeled) or our Triplex labeled RPMI media (RPMI media first prepared without proline, glutamine, or arginine and then supplemented with isotope labeled amino acids at regular RPMI concentrations: 15N-proline (Pro+1; 0.17 mM), 13C,15N-arginine (Arg+2; 0.38 mM), and 13C5-gluatmine (Gln+5; 2.05 mM)). Parasites were incubated in labeled or unlabeled media for 10h at 37ºC under standard malaria gas mixture. Samples were harvested by centrifugation, washed twice, and then polar metabolites extracted.

Sample Preparation:

Sampleprep ID:	SP004380
Sampleprep Summary:	Each sample was washed twice in PBS and then suspended in 10 μL PBS (Life Technologies, Carlsbad, CA, USA). Polar metabolites were extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (0.2 ng/μL valine-d8 (Sigma Aldrich, St. Louis, MO, USA); and 0.2 ng/μL phenylalanine-d8 (Cambridge Isotope Laboratories, Tewksbury, MA, USA) and stored at −80°C prior to the metabolite profiling assays.

Sampleprep ID:

SP004380

Sampleprep Summary:

Each sample was washed twice in PBS and then suspended in 10 μL PBS (Life Technologies, Carlsbad, CA, USA). Polar metabolites were extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (0.2 ng/μL valine-d8 (Sigma Aldrich, St. Louis, MO, USA); and 0.2 ng/μL phenylalanine-d8 (Cambridge Isotope Laboratories, Tewksbury, MA, USA) and stored at −80°C prior to the metabolite profiling assays.

Chromatography:

Chromatography ID:	CH005334
Chromatography Summary:	Chromatography method 1 (HILIC-POS)
Instrument Name:	Shimadzu Nexera X2
Column Name:	Water Atlantis HILIC (150 x 2.1 mm, 3 μm)
Column Temperature:	30
Flow Gradient:	Chromatographic separation was performed using an isocratic elution at a flow rate of 250 μL/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 1 minute. This was followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes. At 10 minutes, the gradient was returned to initial isocratic conditions (5% mobile phase A) and held until 18 minutes, at which point MS acquisition was stopped. The column was equilibrated with 5% mobile phase A at a flow rate of 400 μL/min for 12 minutes, followed by a reduction to the initial flow rate of 250 μL/min for 2 minutes before the next injection.
Flow Rate:	250 μL/min
Internal Standard:	L-Phenylalanine-d8 (CIL, DLM-372-1), L-Valine-d8 (Sigma, 486027)
Solvent A:	100% Water; 10 mM Ammonium formate; 0.1% Formic acid
Solvent B:	100% Acetonitrile; 0.1% Formic acid
Chromatography Type:	HILIC

Analysis:

Analysis ID:	AN007025
Analysis Type:	MS
Chromatography ID:	CH005334
Num Factors:	2
Num Metabolites:	127
Units:	peak areas