MassIVE MSV000079613

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). Cycloheximide was added to parasite-infected red blood cell cultures to a final concentration of 200 uM, followed by 10 min incubation at 37C. Erythrocytes were then pelleted (4 min at 660 x g) and washed twice in PBS containing 200 uM cycloheximide. After the last wash, pellets were kept on ice and were subsequently lysed by adding 2.2 volumes of lysis buffer (1% [v/v] Igepal CA-360 [Sigma-Aldrich] and 0.5% [w/v] sodium deoxycholate in polysome buffer [400 mM potassium acetate, 25 mM potassium HEPES pH 7.2, 15 mM magnesium acetate, 200 uM cycloheximide, 1 mM DTT, and 1 mM AEBSF]). After 10 min incubation on ice, lysates were centrifuged for 10 min at 20,000 x g at 4C. The clarified lysates were then loaded on top of a sucrose cushion (1 M sucrose in polysome buffer) to concentrate the ribosomes. For large cultures volumes, 20 ml lysate was loaded on top of 6 ml of sucrose cushion in 26 ml polycarbonate ultracentrifuge tubes and then centrifuged for 3 h at 50,000 rpm at 4C in a Type 70 Ti rotor (Beckman Coulter, Brea, CA). For small culture volumes, 4 ml lysate was loaded atop 1.25 ml of sucrose cushion in 5 ml polyallomer ultracentrifuge tubes and then centrifuged for 123 min at 50,000 rpm at 4C in an SW 55 Ti rotor (Beckman Coulter). Ribosome pellets were resuspended in polysome buffer, incubated for at least 30 min at 4C to allow complete ribosome resuspension and centrifuged for 10 min at 12,000 x g at 4C. The ribosome suspension was layered on top of a 4.5 ml continuous linear 15-60% sucrose [w/v] gradient in polysome buffer and centrifuged for 1.5 h at 50,000 rpm at 4C in an SW 55 Ti rotor. Fractions of 400 ul were collected using an UA-5 UV detector and model 185 gradient fractionator (ISCO, Lincoln, NE). For the isolation of cytoplasmic fractions, synchronized parasites cultures were lysed by incubation in 0.15% saponin for 10 min on ice. Parasites were centrifuged at 3,234 x g for 10 min at 4C, and washed three times with PBS. After the last wash, parasites were resuspended in PBS, transferred to a microcentrifuge tube and centrifuged for 5 min at 2,500 x g at 4C. Subsequently, the parasite pellet was resuspended in 1.5X volume of cytoplasmic lysis buffer (0.65% Igepal CA-360, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM AEBSF, and EDTA-free protease inhibitor cocktail [Roche]) and lysed by passing through a 26 G half inch needle fifteen times. Parasite nuclei were centrifuged at 14,000 x g for 15 min at 4C, followed by collection of the supernatant containing the cytoplasmic extract. TCA-precipitated protein pellets were digested with endoproteinase LysC followed by trypsin. Polysome replicate 1 samples and the ring stage control sample were analyzed on a Velos pro ion-trap instrument using the LTQ, while all other samples were analyzed using LTQ only. All samples were run in low resolution mode. Fully automated 10-step chromatography runs were carried out, as described in (Florens and Washburn, 2006). 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 1.26% (range, 0.15 - 2.56%) and 0.09% (range, 0.01 - 0.17%), respectively, for the polysome isolation 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: Polysome-associated proteins ; malaria parasite ; intra-erythrocytic cycle

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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.