Dataset Information

Multiple unbiased approaches identify oxidosqualene cyclase as the molecular target of a promising anti-leishmanial

ABSTRACT: Samples were reduced by the addition of TCEP (25 mM final concentration) and incubated at 37 ºC for 10 min. Alkylation was carried out by the addition of iodoacetamide (25 mM final concentration) and incubated at RT for 1 h in the dark. Samples were then digested by the addition of 1:50 LysC (Wako, Japan) in 100 mM triethylammonium bicarbonate (TEAB) followed by incubation at 37 ºC for 6h and then addition of 1:50 trypsin (Thermo) followed by incubation at 37 ºC overnight. The efficiency of sample digestion efficiency was assessed by MS (LTQ XL, Thermo). The samples were then vacuum-dried and resuspended in 100 mM TEAB (100 µL). Samples were then incubated in their respective Tandem Mass Tag™ (TMT) 10-plex reagents (Thermo) for 1 h at RT with agitation. Reactions were quenched by the addition of 5% hydroxylamine for 15 min and each set of samples (treated and vehicle) were pooled in the same tube and dried overnight. The TMTTM-labelled samples were dried and kept at -85 ºC until further analysis.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Leishmania Donovani Bpk282a1

SUBMITTER: Victoriano Corpas-Lopez

LAB HEAD: Susan Wyllie

PROVIDER: PXD023780 | Pride | 2021-07-18

REPOSITORIES: Pride

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Multiple unbiased approaches identify oxidosqualene cyclase as the molecular target of a promising anti-leishmanial.

Paradela Luciana S LS Wall Richard J RJ Carvalho Sandra S Chemi Giulia G Corpas-Lopez Victoriano V Moynihan Eoin E Bello Davide D Patterson Stephen S Güther Maria Lucia S MLS Fairlamb Alan H AH Ferguson Michael A J MAJ Zuccotto Fabio F Martin Julio J Gilbert Ian H IH Wyllie Susan S

Cell chemical biology 20210309 5

Phenotypic screening identified a benzothiophene compound with activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Using multiple orthogonal approaches, oxidosqualene cyclase (OSC), a key enzyme of sterol biosynthesis, was identified as the target of this racemic compound and its enantiomers. Whole genome sequencing and screening of a genome-wide overexpression library confirmed that OSC gene amplification is associated with resistance to compound 1. Introduction ...[more]

PMID: 33691122

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Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.

Dataset Information

Multiple unbiased approaches identify oxidosqualene cyclase as the molecular target of a promising anti-leishmanial

Publications

Multiple unbiased approaches identify oxidosqualene cyclase as the molecular target of a promising anti-leishmanial.

Similar Datasets

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