MassIVE MSV000079612

Description

The Plasmodium falciparum strain 3D7 was cultured in human O+ erythrocytes at 5% haematocrit as previously described (Trager and Jensen, 1976). Cultures were synchronized twice at ring stage with 5% D-sorbitol treatments performed eight hours apart (Lambros and Vanderberg, 1979). Cultures (8% parasitemia in 5% hematocrit in a total volume of 25 ml) were harvested 48 hours after the first sorbitol treatment (ring stage), and then 18 hours (trophozoite stage) and 36 hours thereafter (schizont stage). Parasites from mixed trophozoite and schizont cultures were extracted by saponin lysis of erythrocytes and were crosslinked on ice by 254 nm UV light for a total of 1,200 J/cm2 with two 2-minute breaks with gentle mixing. Following UV-crosslinking, parasites were washed in PBS and lysed in a Lysis/Binding buffer containing 100 mM Tris-HCl, pH7.5, 500 mM LiCl, 1 mM EDTA, 0.5% LiDS, and 5 mM DTT. Negative control samples were lysed in Lysis/Binding buffer without EDTA, treated with 400 ug of RNase A (Life Technologies) and 10,000 units of RNase T1 (Ambion) for 30 min at 37C, followed by the addition of EDTA to a final concentration of 1 mM. Samples were then allowed to bind to magnetic oligo(dT)25 beads (New England Biolabs) by incubating at room temperature for 1 hour with continuous mixing. The beads were washed twice in wash buffer I (20 mM Tris-HCl, pH7.5, 500 mM LiCl, 1 mM EDTA, 0.1% LiDS, and 5 mM DTT), twice in wash buffer II (20 mM Tris-HCl, pH7.5, 500 mM LiCl, and 1 mM EDTA) and once in low-salt buffer (20 mM Tris-HCl, pH7.5, 200 mM LiCl, and 1 mM EDTA). Proteins were eluted in elution buffer (10 mM Tris-HCl, pH7.5, 2 mM CaCl2, and 50 units of MNase) by incubation for 30 min at 37C, followed the addition of Laemmli buffer and a 10-min incubation at 98C. Proteins were precipitated with 20% trichloroacetic acid (TCA). The resulting pellet was washed once with 10% TCA and twice with cold acetone. TCA-precipitated protein pellet (ca. 50 ug) was solubilized in Tris-HCl pH 8.5 and 8 M Urea. TCEP (Tris(2-Carboxylethyl)-Phosphine Hydrochloride, Pierce) and CAM (Chloroacetamide, Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. The protein suspension was digested overnight at 37C using Endoproteinase Lys-C at 1:50 w/w (Roche). Sample was brought to a final concentration of 2 M urea and 2 mM CaCl2 before performing a second overnight digestion at 37C using Trypsin (Promega) at 1:100 w/w. Formic acid (5% final) was added to stop the reactions. Sample was loaded on split-triple-phase fused-silica microcapillary column (McDonald et al., 2002) and placed in-line with linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with quaternary Agilent 1260 series HPLC. Fully automated 10-step chromatography run (for a total of 20 hours) was carried out, as described in (Florens and Washburn, 2006). 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 dynamic exclusion settings used were as follows: repeat count 2; repeat duration 30s; exclusion list size 500 and exclusion duration 120 sec, while the minimum signal threshold was set to 100. The MS/MS dataset was searched using SEQUEST (Eng et al., 1994) against a database of 72,358 sequences, consisting of 5,487 P. falciparum non-redundant proteins (downloaded from PlasmoDB on 2012-07-12), 30,536 H. sapiens non-redundant proteins (downloaded from NCBI on 2012-08-27), 177 usual contaminants (such as human keratins, IgGs, and proteolytic enzymes), and, to estimate false discovery rates, 36,179 randomized amino acid sequences derived from each non-redundant protein entry. To account for alkylation by CAM, 57 Da were added statically to cysteine residues. To account for the oxidation of methionine residues to methionine sulfoxide, 16Da were added as a differential modification to methionine residue. Peptide/spectrum matches were sorted, selected using DTASelect/CONTRAST (Tabb et al., 2002). Proteins had to be detected by 1 peptide with 2 independent spectra, leading to average FDRs at the protein and spectral levels of 0.45% (range, 0-1.13%) and 0.12% (range, 0-0.33%), respectively for the RNA interactome capture experiments. To estimate relative protein levels and to account for peptides shared between proteins, Normalized Spectral Abundance Factors (dNSAFs) were calculated for each detected protein, as described in (Zhang et al., 2010). [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: RNA binding proteins ; malaria parasite ; intra-erythrocytic cycle ; mRNA capture

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Publications

Bunnik EM, Batugedara G, Saraf A, Prudhomme J, Florens L, Le Roch KG.
The mRNA-bound proteome of the human malaria parasite Plasmodium falciparum.
Genome Biol. 2016;17(1):147. Epub 2016 Jul 5.