Summary of Study ST003741

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 PR002327. The data can be accessed directly via it's Project DOI: 10.21228/M8V24V 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	ST003741
Study Title	A Metabolite-Based Resistance Mechanism Against Malaria
Study Summary	Jaundice is a common clinical presentation of Plasmodium (P.) falciparum malaria, arising from the accumulation of circulating bilirubin. Whether the active production of this lipophilic yellow pigment by biliverdin reductase A (BVRA) represents an adaptive or maladaptive response to Plasmodium infection is not understood. Here we found that the transition of P. falciparum infection from asymptomatic towards symptomatic malaria was associated with a >10-fold reduction in the ratio of circulating unconjugated bilirubin over parasite burden. Genetic deletion of Blvra in mice suppressed bilirubin production and precipitated malaria mortality, owed to higher parasite burden and virulence. Inhibition of bilirubin conjugation by the repression of hepatic UDP glucuronosyltransferase family 1 member A1 (UGT1A1), increased the levels of circulating unconjugated bilirubin and prevented malaria mortality. Unconjugated bilirubin targeted P. falciparum directly inside red blood cells (RBC) to suppress mitochondrion pyrimidine synthesis and inhibit the formation of hemozoin crystals, compromising the parasite´s food vacuole´s capacity to detoxify heme and extract essential amino acids (AA) from hemoglobin. In conclusion, jaundice represents an evolutionary conserved metabolic response to Plasmodium spp. infection that limits malaria severity.
Institute	European Molecular Biology Laboratory
Department	EMBL Heidelberg
Last Name	Drotleff
First Name	Bernhard
Address	Meyerhofstr. 1, Heidelberg, BW, 69117, Germany
Email	bernhard.drotleff@embl.de
Phone	none
Submit Date	2025-02-18
Publications	in preparation
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-03-24
Release Version	1

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Project:

Project ID:	PR002327
Project DOI:	doi: 10.21228/M8V24V
Project Title:	A Metabolite-Based Resistance Mechanism Against Malaria
Project Summary:	Jaundice is a common clinical presentation of Plasmodium (P.) falciparum malaria, arising from the accumulation of circulating bilirubin. Whether the active production of this lipophilic yellow pigment by biliverdin reductase A (BVRA) represents an adaptive or maladaptive response to Plasmodium infection is not understood. Here we found that the transition of P. falciparum infection from asymptomatic towards symptomatic malaria was associated with a >10-fold reduction in the ratio of circulating unconjugated bilirubin over parasite burden. Genetic deletion of Blvra in mice suppressed bilirubin production and precipitated malaria mortality, owed to higher parasite burden and virulence. Inhibition of bilirubin conjugation by the repression of hepatic UDP glucuronosyltransferase family 1 member A1 (UGT1A1), increased the levels of circulating unconjugated bilirubin and prevented malaria mortality. Unconjugated bilirubin targeted P. falciparum directly inside red blood cells (RBC) to suppress mitochondrion pyrimidine synthesis and inhibit the formation of hemozoin crystals, compromising the parasite´s food vacuole´s capacity to detoxify heme and extract essential amino acids (AA) from hemoglobin. In conclusion, jaundice represents an evolutionary conserved metabolic response to Plasmodium spp. infection that limits malaria severity.
Institute:	European Molecular Biology Laboratory
Department:	EMBL Heidelberg
Last Name:	Drotleff
First Name:	Bernhard
Address:	Meyerhofstr. 1, Heidelberg, BW, 69117, Germany
Email:	bernhard.drotleff@embl.de
Phone:	none
Publications:	in preparation
Contributors:	Ana Figueiredo, Sonia Trikha Rastogi, Susana Ramos, Fátima Nogueira, Katherine De Villiers, António G. Gonçalves de Sousa, Lasse Votborg-Novél, Cäcilie von Wedel, Pinkus Tober-Lau, Elisa Jentho, Sara Pagnotta, Miguel Mesquita, Silvia Cardoso, Giulia Bortolussi, Andres Munro, Erin M. Tranfield, Jessica Thibaud, Denise Duarte, Ana Laura Sousa, Sandra N. Pinto, Jamil Kitoko, Ghyslain Mombo-Ngoma, Johannes Mischlinger, Sini Junttila, Marta Alenquer, Maria João Amorim, Chirag Vasavda, Piter J. Bosma, Sara Violante, Bernhard Drotleff, Tiago Paixão, Silvia Portugal, Florian Kurth, Laura L. Elo, Bindu Paul, Rui Martins & Miguel P. Soares

Subject:

Subject ID:	SU003873
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Timepoint	Sample source
SA407978	neg h_Blank_2	Blank	12h	Blank
SA407979	pos h_Blank_3	Blank	12h	Blank
SA407980	pos h_Blank_2	Blank	12h	Blank
SA407981	neg h_Blank_1	Blank	12h	Blank
SA407982	pos h_Blank_1	Blank	12h	Blank
SA407983	neg h_Blank_3	Blank	12h	Blank
SA407984	neg8h_Blank_2	Blank	8h	Blank
SA407985	pos8h_Blank_3	Blank	8h	Blank
SA407986	pos8h_Blank_2	Blank	8h	Blank
SA407987	neg8h_Blank_1	Blank	8h	Blank
SA407988	neg8h_Blank_3	Blank	8h	Blank
SA407989	pos8h_Blank_1	Blank	8h	Blank
SA408030	pos h_RBC-BR_2	non-infected Bilirubin	12h	Red blood cells
SA408031	pos h_RBC-BR_1	non-infected Bilirubin	12h	Red blood cells
SA408032	neg h_RBC-BR_1	non-infected Bilirubin	12h	Red blood cells
SA408033	pos h_RBC-BR_4	non-infected Bilirubin	12h	Red blood cells
SA408034	pos h_RBC-BR_3	non-infected Bilirubin	12h	Red blood cells
SA408035	pos h_RBC-BR_5	non-infected Bilirubin	12h	Red blood cells
SA408036	neg h_RBC-BR_5	non-infected Bilirubin	12h	Red blood cells
SA408037	neg h_RBC-BR_4	non-infected Bilirubin	12h	Red blood cells
SA408038	neg h_RBC-BR_3	non-infected Bilirubin	12h	Red blood cells
SA408039	neg h_RBC-BR_2	non-infected Bilirubin	12h	Red blood cells
SA408040	neg8h_RBC-BR_1	non-infected Bilirubin	8h	Red blood cells
SA408041	neg8h_RBC-BR_2	non-infected Bilirubin	8h	Red blood cells
SA408042	neg8h_RBC-BR_3	non-infected Bilirubin	8h	Red blood cells
SA408043	neg8h_RBC-BR_4	non-infected Bilirubin	8h	Red blood cells
SA408044	neg8h_RBC-BR_5	non-infected Bilirubin	8h	Red blood cells
SA408045	pos8h_RBC-BR_2	non-infected Bilirubin	8h	Red blood cells
SA408046	pos8h_RBC-BR_3	non-infected Bilirubin	8h	Red blood cells
SA408047	pos8h_RBC-BR_4	non-infected Bilirubin	8h	Red blood cells
SA408048	pos8h_RBC-BR_5	non-infected Bilirubin	8h	Red blood cells
SA408049	pos8h_RBC-BR_1	non-infected Bilirubin	8h	Red blood cells
SA408050	pos h_RBC-DMSO_2	non-infected DMSO	12h	Red blood cells
SA408051	pos h_RBC-DMSO_1	non-infected DMSO	12h	Red blood cells
SA408052	pos h_RBC-DMSO_3	non-infected DMSO	12h	Red blood cells
SA408053	pos h_RBC-DMSO_4	non-infected DMSO	12h	Red blood cells
SA408054	pos h_RBC-DMSO_5	non-infected DMSO	12h	Red blood cells
SA408055	neg h_RBC-DMSO_3	non-infected DMSO	12h	Red blood cells
SA408056	neg h_RBC-DMSO_1	non-infected DMSO	12h	Red blood cells
SA408057	neg h_RBC-DMSO_4	non-infected DMSO	12h	Red blood cells
SA408058	neg h_RBC-DMSO_2	non-infected DMSO	12h	Red blood cells
SA408059	neg h_RBC-DMSO_5	non-infected DMSO	12h	Red blood cells
SA408060	pos8h_RBC-DMSO_5	non-infected DMSO	8h	Red blood cells
SA408061	pos8h_RBC-DMSO_4	non-infected DMSO	8h	Red blood cells
SA408062	pos8h_RBC-DMSO_1	non-infected DMSO	8h	Red blood cells
SA408063	pos8h_RBC-DMSO_2	non-infected DMSO	8h	Red blood cells
SA408064	pos8h_RBC-DMSO_3	non-infected DMSO	8h	Red blood cells
SA408065	neg8h_RBC-DMSO_5	non-infected DMSO	8h	Red blood cells
SA408066	neg8h_RBC-DMSO_4	non-infected DMSO	8h	Red blood cells
SA408067	neg8h_RBC-DMSO_3	non-infected DMSO	8h	Red blood cells
SA408068	neg8h_RBC-DMSO_1	non-infected DMSO	8h	Red blood cells
SA408069	neg8h_RBC-DMSO_2	non-infected DMSO	8h	Red blood cells
SA407990	neg h_iRBC-BR_4	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407991	pos h_iRBC-BR_1	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407992	pos h_iRBC-BR_3	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407993	neg h_iRBC-BR_5	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407994	pos h_iRBC-BR_2	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407995	neg h_iRBC-BR_3	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407996	neg h_iRBC-BR_2	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407997	neg h_iRBC-BR_1	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407998	pos h_iRBC-BR_4	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA407999	pos h_iRBC-BR_5	Plasmodium falciparum infected Bilirubin	12h	Red blood cells
SA408000	neg8h_iRBC-BR_1	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408001	pos8h_iRBC-BR_3	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408002	pos8h_iRBC-BR_4	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408003	pos8h_iRBC-BR_5	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408004	pos8h_iRBC-BR_2	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408005	neg8h_iRBC-BR_5	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408006	neg8h_iRBC-BR_4	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408007	neg8h_iRBC-BR_3	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408008	neg8h_iRBC-BR_2	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408009	pos8h_iRBC-BR_1	Plasmodium falciparum infected Bilirubin	8h	Red blood cells
SA408010	pos h_iRBC-DMSO_1	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408011	neg h_iRBC-DMSO_3	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408012	pos h_iRBC-DMSO_5	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408013	neg h_iRBC-DMSO_5	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408014	neg h_iRBC-DMSO_4	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408015	pos h_iRBC-DMSO_2	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408016	neg h_iRBC-DMSO_2	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408017	neg h_iRBC-DMSO_1	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408018	pos h_iRBC-DMSO_3	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408019	pos h_iRBC-DMSO_4	Plasmodium falciparum infected DMSO	12h	Red blood cells
SA408020	pos8h_iRBC-DMSO_5	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408021	pos8h_iRBC-DMSO_4	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408022	pos8h_iRBC-DMSO_3	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408023	pos8h_iRBC-DMSO_2	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408024	neg8h_iRBC-DMSO_4	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408025	pos8h_iRBC-DMSO_1	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408026	neg8h_iRBC-DMSO_5	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408027	neg8h_iRBC-DMSO_3	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408028	neg8h_iRBC-DMSO_2	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408029	neg8h_iRBC-DMSO_1	Plasmodium falciparum infected DMSO	8h	Red blood cells
SA408070	negQC09	pooled QC	pooled QC	pooled QC
SA408071	posQC12	pooled QC	pooled QC	pooled QC
SA408072	negQC04	pooled QC	pooled QC	pooled QC
SA408073	negQC11	pooled QC	pooled QC	pooled QC
SA408074	negQC12	pooled QC	pooled QC	pooled QC
SA408075	negQC08	pooled QC	pooled QC	pooled QC
SA408076	negQC07	pooled QC	pooled QC	pooled QC
SA408077	negQC06	pooled QC	pooled QC	pooled QC

Showing page 1 of 2 Results: 1 2 Next Showing results 1 to 100 of 112

Collection:

Collection ID:	CO003866
Collection Summary:	PfD7-GFP cultures (Plasmodium falciparum) were synchronized as described in DOI: 10.1016/j.ejmech.2015.07.047. Trophozoite stage parasites (approximately 18-20h after invasion, 15% parasitemia, 5% HCT, 2 ml culture per sample) were treated with vehicle (0.35% vol/vol DMSO) or bilirubin (41 µM) for 8 or 12h.
Sample Type:	Red blood cells

Treatment:

Treatment ID:	TR003882
Treatment Summary:	PfD7-GFP cultures (Plasmodium falciparum) were synchronized as described in DOI: 10.1016/j.ejmech.2015.07.047. Trophozoite stage parasites (approximately 18-20h after invasion, 15% parasitemia, 5% HCT, 2 ml culture per sample) were treated with vehicle (0.35% vol/vol DMSO) or bilirubin (41 µM) for 8 or 12h.

Sample Preparation:

Sampleprep ID:	SP003879
Sampleprep Summary:	The chemicals used were LC-MS grade water, acetonitrile (ACN), methanol (MeOH), and isopropanol (IPA), which were obtained from Th. Geyer (Germany). High-purity methyl tert-butyl ether (MTBE), ammonium formate, formic acid, ammonium acetate, and acetic acid were purchased from Merck (Germany). Stable isotope labelled internal standards for lipidomics (EquiSPLASH; Avanti Polar Lipids, AL, USA) and metabolomics (MSK-A2-1.2; Cambridge Isotope Laboratories, MA, USA) were used at final concentrations of 0.5% and 1.0% (vol/vol), respectively. The culture medium was completely aspirated and the cells washed with 1XPBS (500xg, 5 min). For biphasic extraction of lipids and polar metabolites, samples were initially quenched by incubation on dry ice with 400 μL of 75% (vol/vol) cold methanol for 20 min and the appropriate internal standards (0.5 µL each/sample) were added. After incubation, the samples were vortexed for 5 min at maximum speed, lysed using an ultrasonic bath to sonicate the sample for 5 min and vortexed again briefly after sonication. After addition of 1000 µL of cold MTBE, the monophasic mixture was vortexed for 60 s and incubated at -20°C for 20 min. For phase separation, 250 µL of cold water were added, followed by another vortexing and incubation step (see previous conditions). The biphasic solvent system was then centrifuged for 15 min at 14,000g and 4 °C. For metabolomics analysis, 400 µL of the lower bottom aqueous phase were transferred, dried under a stream of nitrogen, and reconstituted in 75 µL 80% MeOH (v/v). The final samples were vortexed for 10 min, centrifuged (see previous conditions) and the supernatants were transferred to analytical glass vials for LC-MS/MS analysis.

Sampleprep ID:

SP003879

Sampleprep Summary:

The chemicals used were LC-MS grade water, acetonitrile (ACN), methanol (MeOH), and isopropanol (IPA), which were obtained from Th. Geyer (Germany). High-purity methyl tert-butyl ether (MTBE), ammonium formate, formic acid, ammonium acetate, and acetic acid were purchased from Merck (Germany). Stable isotope labelled internal standards for lipidomics (EquiSPLASH; Avanti Polar Lipids, AL, USA) and metabolomics (MSK-A2-1.2; Cambridge Isotope Laboratories, MA, USA) were used at final concentrations of 0.5% and 1.0% (vol/vol), respectively. The culture medium was completely aspirated and the cells washed with 1XPBS (500xg, 5 min). For biphasic extraction of lipids and polar metabolites, samples were initially quenched by incubation on dry ice with 400 μL of 75% (vol/vol) cold methanol for 20 min and the appropriate internal standards (0.5 µL each/sample) were added. After incubation, the samples were vortexed for 5 min at maximum speed, lysed using an ultrasonic bath to sonicate the sample for 5 min and vortexed again briefly after sonication. After addition of 1000 µL of cold MTBE, the monophasic mixture was vortexed for 60 s and incubated at -20°C for 20 min. For phase separation, 250 µL of cold water were added, followed by another vortexing and incubation step (see previous conditions). The biphasic solvent system was then centrifuged for 15 min at 14,000g and 4 °C. For metabolomics analysis, 400 µL of the lower bottom aqueous phase were transferred, dried under a stream of nitrogen, and reconstituted in 75 µL 80% MeOH (v/v). The final samples were vortexed for 10 min, centrifuged (see previous conditions) and the supernatants were transferred to analytical glass vials for LC-MS/MS analysis.

Combined analysis:

Analysis ID	AN006142	AN006143
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Waters Atlantis Premier BEH Z-HILIC (100 x 2.1mm, 1.7um)	Waters Atlantis Premier BEH Z-HILIC (100 x 2.1mm, 1.7um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Orbitrap Exploris 240	Thermo Orbitrap Exploris 240
Ion Mode	NEGATIVE	POSITIVE
Units	Peak intensity (cps)	Peak intensity (cps)

Chromatography:

Chromatography ID:	CH004665
Chromatography Summary:	LC-MS/MS analysis was performed on a Vanquish UHPLC system coupled to an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA) in negative and positive ESI (electrospray ionization) mode. Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC column (Waters, MA, USA; 2.1 mm x 100 mm, 1.7 µm) at a flow rate of 0.25 mL/min. The mobile phase consisted of water:acetonitrile (9:1, v/v; mobile phase A) and acetonitrile:water (9:1, v/v; mobile phase B), which were modified with a total buffer concentration of 10 mM ammonium acetate, when analysing in negative mode, and 10 mM ammonium formate, when analysing in positive mode. The aqueous portion of each mobile phase was pH-adjusted (negative mode: pH 9.0 via addition of ammonium hydroxide; positive mode: pH 3.0 via addition of formic acid). The following gradient (20 min total run time including re-equilibration) was applied (time [min]/%B): 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95. Column temperature was maintained at 40 °C, the autosampler was set to 4 °C and sample injection volume was 5 µL.
Instrument Name:	Thermo Vanquish
Column Name:	Waters Atlantis Premier BEH Z-HILIC (100 x 2.1mm, 1.7um)
Column Temperature:	40
Flow Gradient:	time [min]/%B: 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95
Flow Rate:	0.25mL/min
Solvent A:	90% water/10% acetonitrile; 10mM ammonium acetate
Solvent B:	90% acetonitrile/10% water; 10mM ammonium acetate
Chromatography Type:	HILIC

Chromatography ID:	CH004666
Chromatography Summary:	LC-MS/MS analysis was performed on a Vanquish UHPLC system coupled to an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA) in negative and positive ESI (electrospray ionization) mode. Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC column (Waters, MA, USA; 2.1 mm x 100 mm, 1.7 µm) at a flow rate of 0.25 mL/min. The mobile phase consisted of water:acetonitrile (9:1, v/v; mobile phase A) and acetonitrile:water (9:1, v/v; mobile phase B), which were modified with a total buffer concentration of 10 mM ammonium acetate, when analysing in negative mode, and 10 mM ammonium formate, when analysing in positive mode. The aqueous portion of each mobile phase was pH-adjusted (negative mode: pH 9.0 via addition of ammonium hydroxide; positive mode: pH 3.0 via addition of formic acid). The following gradient (20 min total run time including re-equilibration) was applied (time [min]/%B): 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95. Column temperature was maintained at 40 °C, the autosampler was set to 4 °C and sample injection volume was 5 µL.
Instrument Name:	Thermo Vanquish
Column Name:	Waters Atlantis Premier BEH Z-HILIC (100 x 2.1mm, 1.7um)
Column Temperature:	40
Flow Gradient:	time [min]/%B: 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95
Flow Rate:	0.25mL/min
Solvent A:	90% water/10% acetonitrile; 10 mM ammonium formate pH3
Solvent B:	90% acetonitrile/10% water; 10 mM ammonium formate pH3
Chromatography Type:	HILIC

MS:

MS ID:	MS005848
Analysis ID:	AN006142
Instrument Name:	Thermo Orbitrap Exploris 240
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Analytes were recorded via a full scan with a mass resolving power of 120,000 over a mass range from 60 – 900 m/z (scan time: 100 ms, RF lens: 70%). To obtain MS/MS fragment spectra, data dependent acquisition was carried out (resolving power: 15,000; scan time: 22 ms; stepped collision energies [%]: 30/50/70; cycle time: 900 ms). Ion source parameters were set to the following values: spray voltage: -3500 V (negative mode), sheath gas: 30 psi, auxiliary gas: 5 psi, sweep gas: 0 psi, ion transfer tube temperature: 350 °C, vaporizer temperature: 300 °C.
Ion Mode:	NEGATIVE

MS ID:	MS005849
Analysis ID:	AN006143
Instrument Name:	Thermo Orbitrap Exploris 240
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Analytes were recorded via a full scan with a mass resolving power of 120,000 over a mass range from 60 – 900 m/z (scan time: 100 ms, RF lens: 70%). To obtain MS/MS fragment spectra, data dependent acquisition was carried out (resolving power: 15,000; scan time: 22 ms; stepped collision energies [%]: 30/50/70; cycle time: 900 ms). Ion source parameters were set to the following values: spray voltage: 4100 V (positive mode), sheath gas: 30 psi, auxiliary gas: 5 psi, sweep gas: 0 psi, ion transfer tube temperature: 350 °C, vaporizer temperature: 300 °C.
Ion Mode:	POSITIVE