Figure 4. Validation of SILAC data by immunoblotting. The following polyclonal antibodies were used: rabbit-anti-PfTrxR (thior- edoxin 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). Densito- metric quantification was performed using the Quantity One software (BioRad) by quantitating band volumina (trace intensity6mm2) of each sample and comparing treated and control band volumina. doi:10.1371/journal.pone.0004098.g004 an upregulation by a factor of 1.6 in the CQ and 1.44 in the artemisinin SIL quantification as well as 1.57 and 1.40 in the immunoblot validation, respectively. Also histone H3, which was quantified in the SIL-MS analysis with 7 (CQ) and 8 (Arte) peptides as upregulated by 1.26 (CQ) and 1.96 (Arte), showed a similar regulation in the immunoblots with values of 1.15 (CQ) and 2.06 (Arte). These experiments prove that the quantifications determined by SIL deliver essentially the same results as the immunoblots. Thus our SIL approach is capable of presenting valuable protein profiling data for more than 800 proteins per comparison. Regulation of proteins under drug pressure Our treatment regimen was to expose the cells in the early trophozoite stage to double IC50–values of the respective drug for twelve hours. An analysis of cell viability after certain time points showed that treated cells were fully viable and proved full reproductive abilities after treatment (see Methods for details). This information is important to verify that the observed proteome changes represent a specific drug response rather than changes induced by cell death. The SIL quantification data show that a fraction of proteins was significantly regulated after treatment with chloroquine or artemisinin under chosen conditions (Table S1 and Table 2). When using a value of 0.5 for down regulation and of 1.5 for upregulation, we observed the following numbers. From 889 quantified proteins in chloroquine treated cells 41 were upregu- lated and 14 down regulated. In artemisinin treated cells, we observed 38 upregulated and 8 down regulated proteins from 804 quantified. These numbers, of course, vary with different set-offs for regulation factors used. Here we chose stringent factors that produce a manageable number of proteins. Table 2 contains selected proteins regulated only under one treatment regimen (chloroquine or artemisinin) or equally regulated under both treatments. Proteins regulated only after chloroquine treatment include for example the conserved protein PFC0135c and the DNA replication licensing factor mcm5, which were both upregulated by more than fivefold and quantified with Drug Induced Proteome Changes five or four peptides, respectively. These proteins were not upregulated under artemisinin treatment; rather, PFC0135C was markedly down regulated (0.28). Overall, sixteen proteins (with six hypothetical and conserved proteins) were specifically upregulated in chloroquine treated cells; six proteins were specifically down regulated. These include four hypothetical proteins and the basic transcription factor 3b. Proteins specifically upregulated (.1.5) in artemisinin treated cells include 18 proteins (with seven hypothetical proteins). These comprise e.g. three proteasomal subunits, a thymidylate kinase and a long-chain fatty acid CoA ligase. Artemisinin treatment led to a reduced expression of five proteins, which include a hypothetical and a conserved protein, a eukaryotic translation initiation factor, a putative glutamate dehydrogenase and the putative co- chaperone GrpE. Interestingly, the conserved protein PFC0135c was down regulated after artemisinin treatment by a factor of 0.28, whereas it was upregulated under chloroquine pressure more than fivefold, as described above. Table 2 furthermore contains proteins which were up- or down regulated similarly under both treatment regimens. Twenty-six proteins were upregulated under both drugs (.1.3). These proteins include histones H3 and H4, the lipoamide dehydroge- nase, a steroid dehydrogenase and a multidrug resistance protein. Again, a huge proportion of proteins (10 out of 26) has not been assigned a function yet. Twenty proteins were down regulated by a factor of at least 0.75 in both samples. To this group eleven hypothetical proteins are counted as well as a putative calcyclin binding protein, a GTP cyclohydrolase I, the ribosomal protein S26e or a putative ubiquitin-conjugating enzyme. 750 proteins were observed in both studies (CQ and arte) and 680 of these showed the same trend (less than 20% difference among the two ratios) in both samples. The rest of the proteins displaying different regulations under artemisinin and chloroquine treatment, as summarized in Table 2, are of special interest as they should represent a drug specific response. Discussion The first large-scale comparative study on the effects of treatment with the antimalarial drugs artemisinin and chloroquine on the proteome of P. falciparum was performed using metabolic labeling followed by identification and quantification with MudPIT LC-MS. The strategy of labeling proteins with stable isotope labeled isoleucine as described by Nirmalan et al. [7] for 2DE was used to create internal standards. By replacing the 2DE procedure with high-resolution LC-MS, where separation, iden- tification and quantification are performed in parallel, we were able to identify and quantify a significantly larger number of proteins. To establish the method described here, the efficiency of labeling parasite proteins with 13C615N1 isoleucine in cell culture was optimized until it reached 99%. Different methods were compared to fractionate cell extracts into sub-proteomes, including weak anion exchange chromatography with salt elution or pH elution and a sequential ammonium sulfate precipitation. As determined by direct comparison of fractionated and unfraction- ated samples, the fractionation procedure described in the methods section enhanced the number of identified proteins by 30%. Combining sample fractionation with the use of a high mass accuracy Orbitrap enabled the identification of an additional 384 proteins that had not been determined previously in proteome analyses. More than 800 proteins could be quantitatively compared between drug treated and untreated samples. Only 100 mg of protein was required for each LC-MS analysis of Plasmodia; thus the method is likely to be applicable for studying PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e4098