free parasitotoxic heme, intercalation with DNA and possible interference with DNA excision repair [13]. More recently, studying the effects of CQ on endocytosis of Plasmodium falciparum revealed an inhibition of vesicle trafficking [14]. As reported by Radfar et al., CQ furthermore mediates specific proteome oxidative damage in the parasite [15]. As suggested by these independent results, CQ may actually act on multiple molecular targets in parallel, which could explain its extraordinary effectiveness over a long period of time. In our study, most proteins showing increases in expression are localized to the nucleus or interact with chromosomes. Also proteins involved in translation show obvious quantitative expression changes. The amplitude of change remains low and variation in specific functional cascades is difficult to characterize, as already reported [16,17]. One of the largest hindrances to identifying particular pathways affected by the drug is the high number of hypothetical proteins that show changes. These proteins can currently not be classified to a particular pathway, which will, however, improve over time. In addition, our data is in accordance with the observation of Gunasekera et al., who observed broad mRNA expression changes in parasites treated with CQ [16,17]. These changes affected particularly ribosomal proteins, signaling molecules, protein processing (heat shock proteins, proteasome subunits, cyclophilin), as well as RNA metabolism (transcription factors). There is a high degree of overlap between the functional group of proteins in the transcriptome data and our proteome study, however, the observed changes are less pronounced at the protein level. A direct and quantitative comparison of the proteome and transcriptome datasets has its limitations since parasite stages employed, synchronicity, drug doses and exposure times varied between the studies. Effects of artemisinin Over the last years artemisinin has become indispensable as an alternative treatment of malaria as the CQ and the sulfadoxine/ pyrimethamine combination have become increasingly ineffective [18]. As for the quinolines many hypotheses for the mechanism of action of artemisinin have been proposed [13,19]. A key feature of all artemisinins is the 1,2,4-trioxane structure with its endoperoxide, which is essential for antimalarial activity. Active endoperoxides are thought to interact with reduced hemin or other sources of ferrous iron inside the parasite forming cytotoxic carbon-centered radical intermediates. These intermediates can alkylate biomolecules like lipids, heme and parasite enzymes including the ATP-dependent Ca2+ pump located on the endoplasmic reticulum, PfATP6. Indeed, our data shows changes of the vacuolar ATP synthase; subunits a and c (PF13_0130) display down regulation when the parasite is treated with artemisinin. In addition, several processes show slight upregulation under artemisinin when classifying the data with GO annotations. This upregulation affects mainly nucleotide and nucleic acid metabolism, transport and secretion as well as the expected response to stimuli. One of the most important observations is that the multidrug resistance gene was found to be upregulated under CQ and artemisinin indicating that pfmdr1 indeed mediates resistance to a number of unrelated classes of agents [20]. Given the fact that approximately 40% of the identified and regulated proteins are hypothetical proteins with unknown functions in the parasite, the complexity of understanding the biology of the malaria parasite is illustrated. Our study is the first to dissect the protein expression response of chloroquine and artemisinin treated Plasmodium falciparum cells on the proteome level. We gain a list of sets of proteins specifically regulated under each of the two drugs and sets of proteins similarly regulated. Thus, our studies represent a basis for quantitative assessment of Drug Induced Proteome Changes protein changes induced by drug treatment. Using this method, further investigations including different drug concentrations and time points in combination with cell biological parameters will allow to further understand mechanisms of action of single drugs or drug combinations. Methods Cultivation of Plasmodium falciparum The chloroquine sensitive P. falciparum strain 3D7 was grown in continuous culture with slight modifications as described before [21]. The 3D7 strain is an established reference strain which had been employed in most of the previous proteomic and transcriptomic studies [9,10]. Briefly, parasites were maintained at 1–10% parasitaemia and 3.3% haematocrit in RPMI 1640 culture medium supplemented with A+ erythrocytes, 4% A+ human serum, 0.2% lipid-rich bovine serum albumin (Albumax), 0.16% glucose, 0.2 mM hypoxanthine, 2.1 mM L-glutamine, and 22 mg/ml gentamycin. Cultures were kept at 37uC, 3% O2, 3% CO2 and 94% N2. Synchronization of parasites in culture to ring stages was carried out by repetitive treatment with 5% (w/v) sorbitol. Parasite growth and parasitaemia were monitored by assessing Giemsa-stained blood smears under the microscope. For determining IC50 and IC90 values on the parasites the semi-automated microdilution technique based on 3H-hypoxanthine incorporation was applied. For immunoblot- ting analyses we prepared biological replicates without SILAC and treated cells equally as in the SILAC experiment. Heavy isotope labeling of P. falciparum proteins For the drug exposure and heavy isotope labeling, specialized cell culture conditions were applied. For this purpose we combined the procedures published by Nirmalan et al., 2004 [7] and Koncarevic et al., 2007 [21]. In contrast to standard conditions, parasites were kept in the absence of human serum, which was replaced by Albumax only. For heavy isotope labeling studies, custom RPMI- 1640 medium devoid of isoleucine was supplemented with 13C6,15N1-isoleucine ($98% isotopic purity; Cambridge Isotope Laboratories, Andover, MA, USA) to yield 7 Da mass shifts per isoleucine in a peptide. Synchronized cultures were preincubated for 24 h in isoleucine free medium before 0.38 mM heavy (13C6,15N1) isoleucine (52.7 mg/ml) or light isoleucine (50 mg/ml) was added to the parallel cultures. Isoleucine incorporation was applied for three complete life cycles (3648 h) before addition of the drugs. Blood stage cultures with a parasitemia of 8–10% at the late ring/early trophozoite stage (24–28 h post infection) were incubat- ed with chloroquine (CQ) or artemisinin (Arte) (26IC50 values = 17 nM for CQ and 35 nM for Arte, determined 72 hrs after addition of a drug which is not removed from the cell culture) or the used solvent (RPMI-1640 medium or DMSO) for control cultures. This experimental setup was based (a) on pretesting of stage specificity and drug concentrations in our lab (b) on the fact that it was desirable to harvest mature trophozoites in order to obtain enough protein for the proteome analyses, and (c) on the available literature which indicates that maximum effects of the two drugs occurred at the late ring and early trophozoite stage, which corresponds to the time at which the most rapid increases in synthetic and glycolytic activities occur. Mature schizonts and young rings are less affected by the antimalarial drugs [9,10]. This data was recently substantiated by Maerki et al. [11] who confirmed in their experiments that trophozoites were more susceptible to CQ than rings or schizonts and that also for artemisinin good effects on trophozoites were determined after 12 h. For each drug, four samples were run in parallel: labeled control, unlabeled control, labeled sample, and unlabeled sample. PLoS ONE | www.plosone.org 7 December 2008 | Volume 3 | Issue 12 | e4098