Large-Scale Differential Proteome Analysis in Plasmodium falciparum under Drug Treatment Judith Helena Prieto1., Sasa Koncarevic2., Sung Kyu Park1, John Yates, III1*, Katja Becker2* 1 Department of Cell Biology, The Scripps Research Institute, La Jolla, California, United States of America, 2 Interdisciplinary Research Center, Justus-Liebig-University, Giessen, Germany Abstract Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action. For most antimalarial drugs neither mode of action nor mechanisms of resistance development are fully elucidated although this would be important prerequisites for successfully developing urgently required novel antimalarials. Here, we establish a large-scale quantitative proteomic approach to examine protein expression changes in trophozoite stages of the malarial parasite Plasmodium falciparum following chloroquine and artemisinin treatment. For this purpose SIL (stable isotope labeling) using 14N-isoleucine and 13C6,15N1-isoleucine was optimized to obtain 99% atomic percent enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS and quantified using the novel software tool Census. In drug treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins identified in the trophozoite stage by 30%. A relative quantification was obtained for more than 800 proteins. Under artemisinin and chloroquine treatment 41 and 38 proteins respectively were upregulated (.1.5) whereas 14 and 8 proteins were down-regulated (,0.5). Apart from specifically regulated proteins we also identified sets of proteins which were regulated as a general response to drug treatment. The proteomic data was confirmed by Western blotting. The methodology described here allows for the efficient large-scale differential proteome analysis of P. falciparum to study the response to drug treatment or environmental changes. Only 100 mg of protein is required for the analysis suggesting that the method can also be transferred to other apicomplexan parasites. Citation: Prieto JH, Koncarevic S, Park SK, Yates J III, Becker K (2008) Large-Scale Differential Proteome Analysis in Plasmodium falciparum under Drug Treatment. PLoS ONE 3(12): e4098. doi:10.1371/journal.pone.0004098 Editor: Debbie Fox, The Research Institute for Children at Children’s Hospital New Orleans, United States of America Received September 22, 2008; Accepted November 21, 2008; Published December 31, 2008 Copyright: 2008 Prieto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was supported by the NIH (AI072615-02), P41 RR011823 and the German Research Council (Be 1540/9-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jyates@scripps.edu (JY); becker.katja@gmx.de (KB) . These authors contributed equally to this work. Introduction The parasitic protozoon Plasmodium falciparum is responsible for approximately 500 million cases of malaria and one million deaths from malaria each year. Recent anti-mosquito measures and new artemisinin-containing treatments prompted calls for global malaria eradication. Novel drugs, vaccines, and insecticides, as well as deeper insights into parasite biology, human immunity, and vector behavior are essential to support these efforts [1]. Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action [2]. Respective analyses identifying proteins of different stages of malarial parasites have been carried out in our and other laboratories [3,4]. Mass spectrometric (MS) methods like the Multidimensional Protein Identification Technology (MudPIT) were developed to enable large scale identification of proteins. In a typical MudPIT analysis an unfractionated protein mixture is digested to peptides, separated by biphasic liquid chromatography (SCX-RP-LC), and analyzed online by tandem mass spectrometry. Such approaches can include either in vitro or in vivo isotope tagging of amino acids which enables pair-wise comparison of protein expression patterns [5,6]. Resulting data provide important insights into molecular mechanisms in cells including stress response and mechanisms of drug action and resistance. Stable isotope labeling with heavy amino acids is a well established technique for protein labeling. It uses metabolic labeling, where the respective isotope-labeled amino acid is translationally inserted into proteins and no chemical labeling and purification steps after labeling are needed. Although stable isotope labeling of Plasmodium is complicated by the required use of red blood cell cultures, the application of SIL was developed for P. falciparum by Nirmalan et al. in 2004 in combination with 2DE [7]. Due to resembling physicochemical properties the isotope labeled proteins or peptides display similar separation characteristics in electrophoresis or chromatography. In liquid-chromatography separation prior to MS the labeled and unlabeled peptides co- elute from the chromatographic column. In the MS they can be resolved, measured, and compared in the same scans. The amino acid best suited for labeling proteins of malaria parasites is isoleucine [4]. It is not synthesized but taken up efficiently by Plasmodium and gives–13C615N1 minus 12C614N1–a spectral sepa- ration of 7.017159 Da. Of all P. falciparum proteins more than 99% have isoleucine containing peptides and thus can be theoretically covered in this approach (plasmodb.org). PLoS ONE | www.plosone.org 1 December 2008 | Volume 3 | Issue 12 | e4098