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Large-Scale Differential Proteome Analysis in Plasmodium falciparum Under Drug Treatment

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Parasite Genomics Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1201))

Abstract

Here, we establish a methodology for large-scale quantitative proteomics using SIL (stable isotope labeling) to examine protein expression changes in trophozoite stages of the malaria parasite Plasmodium falciparum following drug treatment. For this purpose, exposure to 13C6 15N1-isoleucine was optimized in order to obtain 99 % atomic 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 software tool Census. In drug-treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins so far identified in the trophozoite stage by 30 % in the previous literature. A relative quantification was obtained for more than 800 proteins. About 5 % of proteins showed a clear up- or downregulation upon drug treatment.

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References

  1. Gardner MJ, Hall N, Fung E et al (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419:498–511

    Article  CAS  PubMed  Google Scholar 

  2. Florens L, Washburn MP, Raine JD et al (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419:520–526

    Article  CAS  PubMed  Google Scholar 

  3. Winzeler EA (2008) Malaria research in the post-genomic era. Nature 455:751–756

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Lakshmanan V, Rhee KY, Daily JP (2011) Metabolomics and malaria biology. Mol Biochem Parasitol 175:104–111

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. malERA Consultative Group on Basic Science and Enabling Technologies (2011) A research agenda for malaria eradication: basic science and enabling technologies. PLoS Med 8:e1000399

    Article  Google Scholar 

  6. Olsen JV, Schwartz JC, Griep-Raming J et al (2009) A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed. Mol Cell Proteomics 8:2759–2769

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Sharma K, Weber C, Bairlein M et al (2009) Proteomics strategy for quantitative protein interaction profiling in cell extracts. Nat Methods 6:741–744

    Article  CAS  PubMed  Google Scholar 

  8. Park SK, Venable JD, Xu T et al (2008) A quantitative analysis software tool for mass spectrometry-based proteomics. Nat Methods 5:319–322

    PubMed Central  CAS  PubMed  Google Scholar 

  9. Kuss C, Gan CS, Gunalan K et al (2012) Quantitative proteomics reveals new insights into erythrocyte invasion by Plasmodium falciparum. Mol Cell Proteomics 11(M111):010645

    PubMed  Google Scholar 

  10. Dierick JF, Dieu M, Remacle J et al (2002) Proteomics in experimental gerontology. Exp Gerontol 37:721–734

    Article  CAS  PubMed  Google Scholar 

  11. Sanderson SJ, Xia D, Prieto H et al (2008) Determining the protein repertoire of Cryptosporidium parvum sporozoites. Proteomics 8:1398–1414

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Lasonder E, Ishihama Y, Andersen JS et al (2002) Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 419:537–542

    Article  CAS  PubMed  Google Scholar 

  13. Southworth PM, Hyde JE, Sims PF (2011) A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance. Malar J 10:315

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Prieto JH, Koncarevic S, Park SK et al (2008) Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment. PLoS One 3:e4098

    Article  PubMed Central  PubMed  Google Scholar 

  15. Krijgsveld J, Ketting RF, Mahmoudi T et al (2003) Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nat Biotechnol 21:927–931

    Article  CAS  PubMed  Google Scholar 

  16. Wu WW, Wang G, Baek SJ et al (2006) Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF. J Proteome Res 5:651–658

    Article  CAS  PubMed  Google Scholar 

  17. McClatchy DB, Dong MQ, Wu C et al (2007) 15N metabolic labeling of mammalian tissue with slow protein turnover. J Proteome Res 6:2005–2010

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386

    Article  CAS  PubMed  Google Scholar 

  19. Nirmalan N, Sims PF, Hyde JE (2004) Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition. Mol Microbiol 52:1187–1199

    Article  CAS  PubMed  Google Scholar 

  20. Cox J, Matic I, Hilger M et al (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4:698–705

    Article  CAS  PubMed  Google Scholar 

  21. Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193:673–675

    Article  CAS  PubMed  Google Scholar 

  22. Cranmer SL, Magowan C, Liang J et al (1997) An alternative to serum for cultivation of Plasmodium falciparum in vitro. Trans R Soc Trop Med Hyg 91:363–365

    Article  CAS  PubMed  Google Scholar 

  23. Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65:418–420

    Article  CAS  PubMed  Google Scholar 

  24. Fivelman QL, Adagu IS, Warhurst DC (2004) Modified fixed-ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug-resistant strains of Plasmodium falciparum. Antimicrob Agents Chemother 48:4097–4102

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Koncarevic S, Bogumil R, Becker K (2007) SELDI-TOF-MS analysis of chloroquine resistant and sensitive Plasmodium falciparum strains. Proteomics 7:711–721

    Article  CAS  PubMed  Google Scholar 

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Acknowledgement

The authors wish to thank the Alexander von Humboldt Foundation for funding Dr. Judith Helena Prieto. The work was supported by the Deutsche Forschungsgemeinschaft SPP 1710, BE1540/23-1 (to K.B.).

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Correspondence to Katja Becker .

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Prieto, J.H., Fischer, E., Koncarevic, S., Yates, J., Becker, K. (2015). Large-Scale Differential Proteome Analysis in Plasmodium falciparum Under Drug Treatment. In: Peacock, C. (eds) Parasite Genomics Protocols. Methods in Molecular Biology, vol 1201. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1438-8_17

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  • DOI: https://doi.org/10.1007/978-1-4939-1438-8_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1437-1

  • Online ISBN: 978-1-4939-1438-8

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