Metabolic fingerprinting of Buparvaquone and MMV1028806 compared to Atovaquone with 15N Glutamine on T. gondii
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Toxoplasma Gondii (ncbitaxon:5811) Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Martin Blume
PROVIDER: MSV000093447 | MassIVE | Tue Nov 21 07:02:00 GMT 2023
REPOSITORIES: MassIVE
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Project description:HFF cells were infected with PruTom at MOI 1. Intracellular parasites were treated with Atovaquone, Buparvaquone, or MMV1028806 and incubated for three hours with DMEM containing either 25 mM U-13C-glucose or 4 mM 15N-amide-glutamine instead of the respective unlabeled carbon sources. Cultures were quenched in ice-cold PBS and parasites were isolated from their host cells by passaging scraped monolayers through a 23 G needle, filtered through a 3 um polycarbonate filter and after microscopic counting distributed at 1 x 108 parasites per sample. Parasites were washed with ice-cold PBS via 5 min centrifugation at 21'500 x g at 0 degrees. Metabolites were then extracted by sonication for 2 min in 50 uL 80 percent acetonitrile in water. The insoluble particles were pelleted for 5 min at 21,500 x g at 0 degrees. 5 uL of each sample was collected to generate a pooled biological quality control (PBQC) which was injected regularly into the otherwise randomized run list. 5 uL of each sample was injected onto either an ACQUITY UPLC BEH amide column (Waters) equipped with a VanGuard amide pre-column (Waters) or onto a SeQuant ZIC-pHILIC (Merck) column with an OPTI-LYNX ZIC-pHILIC guard column cartridge (Optimize Technologies) operating on a Vanquish Flex (Thermo Fisher). Analytes were chromatographically separated starting with 100 percent eluent A (90 percent acetonitrile in water with 10 mM ammonium carbonate) and 0 percent eluent B (10 mM ammonium carbonate in water), decreasing to 40percent A and 60percent B over 25 min. Metabolites were detected on a Q Exactive Plus (Thermo Fisher) equipped with an electrospray ionization source. To increase coverage of the isotopologues, metabolites were measured in positive and negative mode separately without MS2 scans but with an increased MS1 scan resolution of 140k.
2023-11-21 | MSV000093445 | MassIVE
Project description:HFF cells were infected with PruTom at MOI 1. Approximately 64 hours later, the cultures were treated with 1.5 uM MMV019189, 7 uM MMV1028806, 30 uM MMV228911, 1.2 uM MMV595321, 5 uM MMV688762, 2 uM MMV688854,respectively, and 0.1 percent DMSO as a control for 3 hours. These concentrations correspond to the five-fold respective IC50 values. Cultures were quenched in ice-cold PBS and parasites were isolated from their host cells by passaging scraped monolayers through a 23 G needle, filtered through a 3 um polycarbonate filter and after microscopic counting distributed at 1 x 108 parasites per sample. Parasites were washed with ice-cold PBS via 5 min centrifugation at 21'500 x g at 0 degrees. Metabolites were then extracted by sonication for 2 min in 50 uL 80 percent acetonitrile in water. The insoluble particles were pelleted for 5 min at 21,500 x g at 0 degrees. 5 uL of each sample was collected to generate a pooled biological quality control (PBQC) which was injected regularly into the otherwise randomized run list. 5 uL of each sample was injected onto either an ACQUITY UPLC BEH amide column (Waters) equipped with a VanGuard amide pre-column (Waters) or onto a SeQuant ZIC-pHILIC (Merck) column with an OPTI-LYNX ZIC-pHILIC guard column cartridge (Optimize Technologies) operating on a Vanquish Flex (Thermo Fisher). Analytes were chromatographically separated starting with 100 percent eluent A (90 percent acetonitrile in water with 10 mM ammonium carbonate) and 0 percent eluent B (10 mM ammonium carbonate in water), decreasing to 40percent A and 60percent B over 25 min. Metabolites were detected on a Q Exactive Plus (Thermo Fisher) equipped with an electrospray ionization source operated in rapid polarity-switching mode at an MS1 resolution of 70k and an MS2 resolution of 35k (Top3 MSX1).
2023-11-21 | MSV000093444 | MassIVE
Project description:We reported that the microRNA profiling of exosome isolated from 58 cerebrospinal fluid (CSF) of 38 medulloblastoma (MBL) patient with or without letomeningeal metastases (LM). CSF exosome was precipitated using Exo2DTM for RNA assay (EXOSOME plus, Suwon, South Korea) according to the manufacture’s protocol (Kim, et al. 2017). 2 ml of CSF were added to Exo2DTM reagent B following mixing by inverting, and incubated at room temperature for 30 min. The supernatant was discarded and the exosome containing pellet was resuspended in 100 ul of PBS to further RNA isolation. Total RNA was extracted from the CSF exosome fraction using a microRNA purification kit (Norgen Biotek corp., Thorold, Canada) according to the manufacture’s protocol. Briefly, samples were mixed with lysis buffer, and the lysate was added to absolute ethanol. The lysate was applied up to the column and centrifuged at 8,000 rpm for 1 min. The column was washed with wash solution and centrifuged at 14,000 rpm for 1 min. Finally, the column was applied to 20 ul of elution solution and centrifuged at 14,000 rpm for 2 min, and the flow-through was stored at –80 ℃ for storage. RNA precipitates were quantified and qualified with Bioanalyzer Pico 6000 Chip (Agilent Technologies, Santa Clara, CA). The 10 ng of total RNA was provided to the cDNA library generation using SMARTer smRNA-Seq kit (Illumina Inc., San Diego, CA), according to the user manual. The cDNA library was subjected to sequencing using Truseq SBS Kit and HiSeq 2500 System (Illumina Inc., San Diego, CA), according to user guide.
2020-09-15 | GSE157916 | GEO
Project description:We describe the use of conventional flow proteomics (200 uL/min) on an optimized system for the analysis of highly fractionated TMT labeled materials. We compare 20ug injections on column using NanoFreeProteomics (NFP) to 1uL injections on a convenentional nanoLC system
2020-02-25 | PXD016704 | Pride
Project description:Female Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.
2014-02-14 | PXD000354 | Pride
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The solid phase extraction (SPE) of the ethanol extracts was performed with the Oasis HLB 96-well plate (30 mg Sorbent per Well, 30 um Particle Size) (Waters Corporation) set up with a vacuum pump manifold. Each well of the plate was washed with 700 uL of 100 percent HPLC-grade methanol and equilibrated with 700 uL of HPLC-grade water. Next, 400 uL of ethanol extracts were added to the wells and allowed to slowly elute. Wells were next washed with 800 uL of 5 percent methanol in water. Metabolites from bacterial cultures or control media were eluted with 200 uL of 100 percent methanol. Vacuum up to 20 psi was applied for the wells that did not elute within one hour. The collected eluted metabolites were stored at -20 until the HPLC-MS analysis. The HPLC-MS analysis of extracted metabolites was performed with the use of a Dionex UltiMate 3000 UHPLC system (Fisher Scientific) coupled to a Bruker impact HD quadrupole time-of-flight mass spectrometer (Bruker). The chromatographic separation was performed on a Kinetex C18 1.7 um, 100A UHPLC column (50 mm x 2.1 mm) (Phenomenex), held at 40C during analysis. 5 uL of each sample was injected. Mobile phase A was water, 0.1 percent v:v formic acid, and mobile phase B was acetonitrile, 0.1 percent v:v formic acid. The solvent gradient table was set as follows: 2 percent B, increased to 10 percent B over 0.2 min, then to 100 percent B at 12 min, held at 100 percent B for 1.5 min, decreased back to 2 percent B in 0.5 min, followed by washout cycle and equilibration; total analysis time of 15 min. The scanned m/z range was 80-2000 Th; capillary voltage 4,500 V; nebulizer gas pressure 2 bar, drying gas flow rate 9 l min-1 and temperature 200C. The full MS scan was followed by a tandem mass spectrometry fragmentation of the seven most abundant ions within the spectrum. For tandem mass spectrometry, the collision cell collision energy was set at 3 eV and the collision energy was stepped 50 percent, 75 percent, 150 percent, and 200 percent. The scan rate was 3 Hz. A HP-921 lock mass compound was infused during the analysis for post processing mass correction.
2022-10-17 | MSV000090542 | MassIVE
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2014-01-09 | PXD000658 | Pride
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2024-08-02 | MSV000095508 | MassIVE
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2009-11-10 | GSE18834 | GEO
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2022-08-12 | PXD031560 | Pride