Drug Induced Proteome Changes 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. doi:10.1371/journal.pone.0004098.g002 energy, amino acid, and nucleotide metabolism but also cell communication, transport, and biosynthesis. As deducible from a ranked list of all P. falciparum genes on PlasmoDB, many of these identified proteins represent important antimalarial drug targets. The opportunity to study these proteins quantitatively and differentially in a large scale approach will support future drug development strategies. In accordance with the genome annota- tions, a considerable percentage of the identified proteins are of unknown function. The methodology described here will facilitate more detailed studies. Relative quantification of isoleucine containing proteins Relative quantification of isoleucine containing peptides was performed using the software tool Census [8]. We were able to quantify an average of 75% of all identified isoleucine-containing peptides for each dataset. In the chloroquine treated cells we relatively quantified 889 proteins, in the artemisinin treated cells 814. Figure 3 displays the count of peptides per quantified protein, showing that for quantification of 83% of the proteins at least two peptides were used. The average number of measured peptides per identified protein ranged from ,5.8 to ,6.2 in our experiments. A validation of the quantification data was performed for five proteins for which antibodies were available. The immunoblots for the selected proteins are shown in Figure 4. The parasite proteins thioredoxin reductase (TrxR), glutathione reductase (GR), and thioredoxin (Trx) displayed no significant regulation in the mass spectrometry experiments (Table S1), which was confirmed in the immunoblots. For EBA-75 (erythrocyte binding antigen) we saw 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. doi:10.1371/journal.pone.0004098.g003 PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e4098