Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment.
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 (13)C6 (15)N1-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.
Chemistry Department, Western Connecticut State University, Danbury, CT, USA.
Judith Helena
JH
Prieto
Elisabeth
E
Fischer
Sasa
S
Koncarevic
John
J
Yates
Katja
K
Becker
D018929:Cell Culture Techniques; D005591:Chemical Fractionation / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003470:Culture Media; D020128:Inhibitory Concentration 50; D007532:Isoleucine / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000032:analysis* / Q000302:isolation & purification; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods
D003470:Culture Media; D015800:Protozoan Proteins; D007532:Isoleucine