After 12 hours of drug exposure the parasites had reached the late trophozoite/schizont stage and were harvested in parallel with the controls. The window of the sorbitol-induced synchrony (.90%) at which the trophozoites were harvested was approximately 5 hours. This window was achieved by repetitive sorbitol-induced synchronizations (48 h, 96 h, 144 h, and 192, and 198 h before the ring stage parasites that were later used for the experiment were obtained). Since no reinvasion step was present during drug exposure, the incubation time was rather short and the drug concentrations were rather low (26IC50) the parasitemia did not change within the 12 hours after drug treatment. Parasite viability after drug treatment A control experiment was conducted in order to assess the viability of the parasites after drug exposure. Parasites (6.4% parasitemia) were exposed to the drugs as described above. After 12 h the drugs were removed by changing the medium and washing the cells once in medium. Cultures were then maintained under normal growth conditions. The next schizogony took place simultaneously in the three cultures resulting in 11.6% parasitemia with 5.4% residual schizonts (CQ), in 11.6% parasitemia with 5.1% schizonts (Arte) and in 11.7% parasitemia with 5.0% schizonts at the given window of the sorbitol-induced synchrony. In spite of similar growth behavior, drug treated schizonts appeared slightly denser under the light micro- scope. Cultures were then splitted (1:5 dilution to reduce the high parasitemia) and incubated further. 17 h after peak schizogony parasitemias of 6.3% rings (CQ), 6.4% rings (Arte) and 6.4% (control) were determined. The data indicates that–as intended-the drug treatment protocol applied in our study did only slightly affect the parasites and did not lead to major growth retardation or cell death. Treated and untreated parasites differed by less than 1 hour in their develop- mental stage. In fact, the aim in our study was to distinguish between the changes from halted growth and the direct effect of drug treatment using two drugs that are known to act on trophozoites. The protein expression changes observed under both drugs are likely to reveal the changes observed from halted development whereas the differences are related to the specific action of each drug. By using this method, further investigations using different drug concentra- tions and time points in combination with cell biological parameters will allow a better understanding of mechanisms of action of single drugs or drug combinations. Preparation of parasite extracts The preparation of parasite extracts followed established protocols [21]. Briefly, parasites were isolated by lysing the red blood cell in saponin containing buffer (7 mM K2HPO4, 1 mM NaH2PO4, 11 mM NaHCO3, 58 mM KCl, 56 mM NaCl, 1 mM MgCl2, 14 mM glucose, 0.02 mM saponin, pH 7.5) followed by intensive washing. Parasites were disrupted by three cycles of freezing and thawing and ultrasonication in digestion buffer (4 M urea, 0.4% Triton X-100, 50 mM Tris-HCl, 5 mM EDTA, 10 mM MgSO4, pH 8.0) in the presence of protease inhibitors. RNA/DNA-digestion was performed with benzonaseH (Merck) for 30 min at 4uC. A first centrifugation step at 15,000 g removed contaminating haemozoin. The supernatant after centrifugation at 100,000 g for 30 min was used as ‘‘parasite’’ extract for fractionation. The insoluble pellet was used as ‘‘insoluble fraction’’ for the analyses. Protein immunoblotting analysis The electrophoretic pattern of some proteins was investigated by immunoblot analysis using specific polyclonal antibodies. Proteins were resolved by Tris-Tricine-PAGE and were trans- ferred onto a PVDF membrane. The blots were incubated with Drug Induced Proteome Changes antibodies and washed according to standard procedures. The following polyclonal antibodies were used: rabbit-anti-PfTrxR, rabbit-anti-PfGST, rabbit-anti-PfGR (available in the Becker Lab), rabbit-anti-Histone H3 (Abcam, Cambridge, UK), rabbit-anti- PfEBP1 (MRA-2 by MR4) at appropriate dilutions 1:1,000–5,000 and peroxidase-conjugated anti-rabbit antibody (Dianova, Ham- burg, Germany, anti-IgG, 1:50,000–100,000). Vizualisation was performed by enhanced chemiluminescence (ECL, SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnolo- gy, Rockford, IL, USA). Densitometric quantitation was per- formed using the Quantity One software (BioRad) by quantitating band volumina (trace intensity6mm2) of each sample and comparing treated and control band volumina. Proteome subfractionation To reduce sample complexity (number of protein species/ sample) and to enrich proteins being of ‘‘lower abundance’’ out of the unfractionated samples, we established a procedure for subfractionating the Plasmodium falciparum proteome. Before fractionation, samples to be compared (light and heavy) were mixed in a protein to protein ratio of 1:1 wt/wt. Weak anion exchanger chromatography was used to subfractionate the soluble proteins of P. falciparum. Vivapure Mini H, D ion exchanger columns (Diethylamine (D). Weak basic anion exchanger (R-CH2- NH+-(CH2H5)2, Sartorius, Germany) were loaded with the sample according to the manufacturer’s instructions. The columns were equilibrated with a buffer containing 2 M urea, 0.2% Triton, 25 mM Tris, 2.5 mM EDTA, 5 mM MgSO4, pH 8.0 and then loaded with 1:1 mixed protein extract. Bound proteins were eluted according to two different procedures. The first procedure included elution according to pH (buffer containing 2 M urea, 0.2% Triton, 25 mM Tris (or sodium acetate), 2.5 mM EDTA, 5 mM MgSO4, pH 8.0, 7.0, and 3.0. For effective elution each step was performed with three single column volumina. The second elution procedure was performed by stepwise increasing salt concentrations (200–500 mM NaCl). Furthermore a third procedure for crude sample fractionation was applied by sequential NH4SO4 precipitation (40%, 70%, and 90% NH4SO4). Protein concentration in eluted samples was measured, fractions were precipitated by OrgoSolTM-DETERGENT-OUTTM Deter- gent Removal Kit (Calbiochem, Darmstadt, Germany) and separated by SDS-PAGE to monitor fractionation or used for LC-MS analyses. Multidimensional protein identification technology (MudPIT) Precipitated P. falciparum protein preparations were dissolved in digestion buffer, digested with trypsin and LysC, and analyzed by LC/LC/MS/MS according to published protocols [22,23]. Approximately 100 mg of protein was used for a 6-step for soluble samples and 12-step for insoluble LC/LC/MS/MS analysis on a LTQ-Orbitrap, a hybrid mass spectrometer in which a linear ion trap is coupled to an Orbitrap mass analyzer (ThermoElectron, San Jose, CA). All samples were analyzed in triplicate. The obtained MS/MS spectra were analyzed with SEQUEST 2.7 [24,25] using a non-redundant Plasmodium database (PlasmoDB version 5.0) [26]. The SEQUEST outputs were analyzed by DTASelect 2.0 [27]. DTASelect 2.0 uses a quadratic discriminant analysis to dynamically set XCorr and DeltaCN thresholds for the entire data set to achieve a user-specified false positive rate (5% in this analysis). The false positive rates were estimated by the program from the number and quality of spectral matches to the decoy database [28]. PLoS ONE | www.plosone.org 8 December 2008 | Volume 3 | Issue 12 | e4098