Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment

Abstract

Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action. For most antimalarial drugs neither mode of action nor mechanisms of resistance development are fully elucidated although this would be important prerequisites for successfully developing urgently required novel antimalarials. Here, we establish a large-scale quantitative proteomic approach to examine protein expression changes in trophozoite stages of the malarial parasite Plasmodium falciparum following chloroquine and artemisinin treatment. For this purpose SIL (stable isotope labeling) using (14)N-isoleucine and (13)C(6),(15)N(1)-isoleucine was optimized to obtain 99% atomic percent enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS and quantified using the novel software tool Census. In drug treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins identified in the trophozoite stage by 30%. A relative quantification was obtained for more than 800 proteins. Under artemisinin and chloroquine treatment 41 and 38 proteins respectively were upregulated (>1.5) whereas 14 and 8 proteins were down-regulated (<0.5). Apart from specifically regulated proteins we also identified sets of proteins which were regulated as a general response to drug treatment. The proteomic data was confirmed by Western blotting. The methodology described here allows for the efficient large-scale differential proteome analysis of P. falciparum to study the response to drug treatment or environmental changes. Only 100 microg of protein is required for the analysis suggesting that the method can also be transferred to other apicomplexan parasites.

Figure 1. Quantification improvement by fractionation of cell extracts.

A: Fractionation of P. falciparum trophozoite extracts by centrifugation and low-strength anion exchange chromatography. F1 to F3 are fractions derived by separation of the 100.000 g supernatant on low-strength anion exchanger chromatography. F1 (flowthrough); F2 (200 mM NaCl eluate); F3 (500 mM eluate); F4 (100.000 g pellet). The band pattern shows a fractionation into different subproteomes. B: Venn diagram comparing numbers of protein identifications of a previous MudPIT study by Florens et al. and our current study, where prefractionation was applied. C: Venn diagram summarizing numbers of protein identifications per fraction and overlaps with other fractions.

Figure 2. Functional classification of identified proteins.

Identified proteins were classified using go annotations downloaded from PlasmoDB (www.plasmodb.org) and simplifying it with go-slim. The classification shows a widespread distribution of the identified proteins in metabolism.

Figure 3. Number of peptides per protein used for quantification.

Number of identified peptides with at least one isoleucine in their sequence per protein that have been quantified using Census against their incidence.

Figure 4. Validation of SILAC data by immunoblotting.

The following polyclonal antibodies were used: rabbit-anti-PfTrxR (thioredoxin reductase), rabbit-anti-PfGST (glutathione S-transferase), rabbit-anti-PfGR (glutathione reductase) (available in the Becker Lab), rabbit-anti-Histone H3 (Abcam, Cambridge, UK), rabbit-anti-PfEBA (Erythrocyte Binding Antigen) (MRA-2 by MR4) at appropriate dilutions 1∶1,000–5,000 and peroxidase-conjugated anti-rabbit antibody (Dianova, Hamburg, Germany, anti-IgG, 1∶50,000–100,000). Visualization was performed by enhanced chemiluminescence (ECL, SuperSignal West Femto Maximum Sensitivity Substrate, Pierce Biotechnology, Rockford, IL, USA). Densitometric quantification was performed using the Quantity One software (BioRad) by quantitating band volumina (trace intensity×mm²) of each sample and comparing treated and control band volumina.