Dataset Information

MudPIT analyses of the proteins associated with the CRWN-like protein isolated from mixed stages of Plasmodium falciparum intraerythrocytic development cycle

ABSTRACT: Protein pull-down assay- Mixed erythrocytic stage 3D7 Plasmodium falciparum parasite cultures were collected and lysed using 0.15% saponin for 10 min on ice. After subsequent washes, the parasite pellet was resuspended in 2.5X volume of IP buffer (0.65% Igepal CA-360 (Sigma-Aldrich), 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 1 mM AEBSF, 5 uM E-64 and EDTA-free protease inhibitor cocktail (Roche)) and lysed by passing through a 26G 1/2-inch needle ten times and sonicated 7 times 10 seconds on/30 seconds off using a probe sonicator. Extracted nuclear protein lysates were incubated for 10 mins at room temperature with DNase I to remove DNA and centrifuged for 10 mins at 14,500 rcf to remove cell debris. Washed Protein A magnetic beads (Pure Proteome) were added to the protein sample and incubated for 1 hour at 4oC to preclear the lysate. Precleared lysate was transferred to a new microcentrifuge tube and split equally for the antibody and no antibody control. The anti-CRWN-like custom antibody was added at a 1:50 ratio and incubated overnight at 4oC. The negative control with no antibody was also incubated overnight. Antibody-protein complexes were recovered using Protein A magnetic beads (Pure Proteome), followed by extensive washes with wash buffer A (1% Triton-X, 1 mM EDTA in 1X PBS), wash buffer B (wash buffer A, 0.5 M NaCl) and wash buffer C (1 mM EDTA, 1X PBS). Proteins were eluted using 0.1 M glycine, pH 2.8 and the eluent was neutralized using 2 M Tris-HCl, pH 8.0. Protein eluates were TCA-precipitated overnight, then washed twice with cold acetone. Multidimensional Protein Identification Technology- The TCA-precipitated protein pellet was solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 5,530 P. falciparum non-redundant proteins (PlasmoDB-34), 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 42,351 randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues. To account for the oxidation of methionine to methionine sulfoxide, 16 Da were added as a differential modification to the methionine residue. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.

INSTRUMENT(S): LTQ

ORGANISM(S): Plasmodium Falciparum 3d7 (ncbitaxon:36329)

SUBMITTER: Laurence Florens

PROVIDER: MSV000084671 | MassIVE | Tue Dec 10 11:45:00 GMT 2019

SECONDARY ACCESSION(S): PXD016684

REPOSITORIES: MassIVE

ACCESS DATA

Publications

The chromatin bound proteome of the human malaria parasite.

Batugedara Gayani G Lu Xueqing M XM Saraf Anita A Sardiu Mihaela E ME Cort Anthony A Abel Steven S Prudhomme Jacques J Washburn Michael P MP Florens Laurence L Bunnik Evelien M EM Le Roch Karine G KG

Microbial genomics 20200204 2

Proteins interacting with DNA are fundamental for mediating processes such as gene expression, DNA replication and maintenance of genome integrity. Accumulating evidence suggests that the chromatin of apicomplexan parasites, such as <i>Plasmodium falciparum</i>, is highly organized, and this structure provides an epigenetic mechanism for transcriptional regulation. To investigate how parasite chromatin structure is being regulated, we undertook comparative genomics analysis using 12 distinct euk ...[more]

PMID: 32017676

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Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

Project description:Protein pull-down assay- Mixed erythrocytic stage 3D7 Plasmodium falciparum parasite cultures were collected and lysed using 0.15% saponin for 10 min on ice. After subsequent washes, the parasite pellet was resuspended in 2.5X volume of IP buffer (0.65% Igepal CA-360 (Sigma-Aldrich), 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 1 mM AEBSF, 5 uM E-64 and EDTA-free protease inhibitor cocktail (Roche)) and lysed by passing through a 26G 1/2-inch needle ten times and sonicated 7 times 10 seconds on/30 seconds off using a probe sonicator. Extracted nuclear protein lysates were incubated for 10 mins at room temperature with DNase I to remove DNA and centrifuged for 10 mins at 14,500 rcf to remove cell debris. Washed Protein A magnetic beads (Pure Proteome) were added to the protein sample and incubated for 1 hour at 4oC to preclear the lysate. Precleared lysate was transferred to a new microcentrifuge tube and split equally for the antibody and no antibody control. The anti-SMC3 custom antibody was added at a 1:50 ratio and incubated overnight at 4oC. The negative control with no antibody was also incubated overnight. Antibody-protein complexes were recovered using Protein A magnetic beads (Pure Proteome), followed by extensive washes with wash buffer A (1% Triton-X, 1 mM EDTA in 1X PBS), wash buffer B (wash buffer A, 0.5 M NaCl) and wash buffer C (1 mM EDTA, 1X PBS). Proteins were eluted using 0.1 M glycine, pH 2.8 and the eluent was neutralized using 2 M Tris-HCl, pH 8.0. Multidimensional Protein Identification Technology- Two biological replicates with two technical replicates each were prepared for ChEP and cytoplasmic control samples at the ring, trophozoite, and schizont stages. Proteins were precipitated with 20% trichloroacetic acid (TCA) and the resulting pellet was washed once with 10% TCA and twice with cold acetone. About 50 ug of the TCA-precipitated protein pellet was solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 5,530 P. falciparum non-redundant proteins (PlasmoDB-34), 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 42,351 randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues. To account for the oxidation of methionine to methionine sulfoxide, 16 Da were added as a differential modification to the methionine residue. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.

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Dataset Information

MudPIT analyses of the proteins associated with the CRWN-like protein isolated from mixed stages of Plasmodium falciparum intraerythrocytic development cycle

Publications

The chromatin bound proteome of the human malaria parasite.

Similar Datasets

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