In the present study we aimed at developing a large-scale quantitative proteomic approach in malarial parasites as an efficient method for studying cellular response to e.g. drug pressure or environmental changes. For this purpose we employed isoleucine-based SILAC in combination with proteome fraction- ation via anion exchange chromatography, SCX/RP for peptide separation, LC-MS/MS analysis, and quantification using the novel software tool Census. This strategy represents a general approach that can be used to study the mechanism of action for drug treatment of pathogens. Results Within the framework of this study we established the first large scale comparative proteomics analysis for Plasmodium falciparum. To optimize labeling efficiency with 13C615N1 isoleucine three complete cycles of growth (72 hours) were carried out leading to an average labeling efficiency of 99%. The use of high mass accuracy instruments and new software tools maximized the number of protein identifications, while at the same time enabling quantitative comparisons. Protein Identification The measurement of tryptic digests of P. falciparum trophozoite stages by MudPIT led to the identification of 1,253 parasite proteins (or 6,318 peptides) in total. The numbers of identifications and regulated proteins for the drug-treated samples are shown in Table 1. The supplementary Table S1 contains all identifications from trophozoite extracts produced in this study. The data obtained allowed for a significant enhancement of protein identification numbers for this parasite stage compared to previous studies [3,4]. This improvement was largely supported by a prefractionation step on the protein level. Fractionation of the soluble protein extract was performed on weak anion exchanger chromatographic columns to obtain three different protein fractions. These fractions plus the insoluble 100.000 g protein pellet constitute the four protein fractions used in our MS experiments. SDS-PAGE of the obtained fractions indicates that separation into subfractions was successful as the protein band patterns of the fractions differ considerably (Figure 1a). To define the potential benefit of our fractionation procedure, we compared ID numbers of MudPIT runs of the unfractionated soluble sample to the fractionated soluble sample (three fractions). Through fractionation the overall number of IDs increased by about 30% as well as the quantification of all peptides identified. Figure 1b summarizes the number of protein identifications in non-fractionated and fractionated samples. Of all Plasmodium predicted proteins, all except one contain at least one isoleucine and thus could theoretically be quantified by an approach using labeled isoleucine. Table 1. Total number of proteins identified in trophozoite stages of Plasmodium falciparum after drug treatment. Furthermore the numbers of regulated proteins are given. Chloroquine Artemisinin Total Protein Identifications with Isoleucine Overlap with Control Upregulated (.1.5) Down Regulated (,0.5) doi:10.1371/journal.pone.0004098.t001 1211 889 41 14 1165 814 38 8 Drug Induced Proteome Changes 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. [3] and our current study, where prefractionation was applied. C: Venn diagram summarizing numbers of protein identifications per fraction and overlaps with other fractions. doi:10.1371/journal.pone.0004098.g001 In order to assess the benefit of the presented experimental setup, we submitted the mass spectrometric datasets of a previous study conducted in our lab [3] and the present study to the same search and filtering parameters for a comparable amount of MudPIT runs. We detected an increase in the number of protein identifications of 34% (data not shown). Thus, our study adds 384 proteins to the erythrocytic stage that had not been identified previously [3,4], which represents a further step towards the completion of the Plasmodium falciparum proteome in the erythro- cytic stages. The increase of identifications through fractionation is displayed in the Venn diagram (Figure 1c). Proteins that are exclusively found in the respective fractions are shown. The distribution of the functional profiles of the identified proteins is summarized in Figure 2. Large parts of the measured proteome contribute to important cellular functions including PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e4098