Proteomic Profiling of Plasmodium falciparum through Improved, Semiquantitative Two-Dimensional Gel Electrophoresis

Abstract

Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria parasite) proteome is still limited due to inefficient extraction and detection methods and the extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the most appropriate methods for Plasmodial protein quantification and 2-DE detection, and finally protein identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis buffer was only possible with the 2-D Quant kit, and of the four stains investigated, Flamingo Pink was superior regarding sensitivity, linearity, and excellent MS-compatibility. 2-DE analyses of the Plasmodial proteome using this methodology resulted in the reliable detection of 349 spots and a 95% success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57 and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally highlights the presence of various isoforms within the Plasmodial proteome, which is of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle.

Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum

“…Similar evidence is presented for an intraparasitic localization of human paraoxonase, which had previously been encountered in other proteomic studies of P. falciparum (17,55). An intraparasitic localization of flavin reductase and catalase is also consistent with previous studies (18,22,33,55,56), whereas lactoferrin has-to the best of our knowledge-not been reported to be present in malaria parasites before.…”

“…In fact, flavin reductase has recently also been reported to exhibit increased abundance in the parasite after drug treatment, in this case with doxycycline (18). Furthermore, we and others (33,56) have detected human selenium-binding-protein 1 in parasite preparations which could potentially facilitate the activity of selenocysteine-containing proteins with a redox function such as human glutathione peroxidase or the parasite protein Sel2 (62). In principle, the use of human enzymes by P. falciparum to bolster its antioxidant defense is especially plausible be-cause these parasites lack the genes for both catalase and a genuine glutathione peroxidase (63).…”

Proteomics of the Rodent Malaria Parasite Using Matrix-Assisted Laser Desorption/Ionization Quadrupole Ion Trap Time-of-Flight Tandem Mass Spectrometry

“…In MS/MS measurements, the following precursor ion species were selected: disulfide isomerase, ions at m/z 1346.7, 1733.8, 1868.0, 2522.2, and 2786.3; actin, ions at m/z 1515.8, 1661.8, 1776.9, and 2261.1; RNA helicase-1, ions at m/z 1567.8 and 1843.9; and hypothetical protein, ions at m/z 975.5, 1094.5, and 1728.7. The Mascot search results and the agreement of both Mr and pI values with those of P. faluciparum previously reported 5,[16][17][18] indicated that disulfide isomerase, actin, and RNA helicase-1 identifications in this study were acceptable, while there was disagreement about the Mr value in the hypothetical protein.…”

“…5 2-D PAGE has several advantages such as good resolution of abundant proteins as well as information on relative molecular mass (Mr), quantity, and isoforms with posttranslational modifications of different isoelectric points (pIs). Mass spectral investigation requires soft ionization techniques.…”

“…Mass spectral investigation requires soft ionization techniques. Electrospray ionization using LC-MS/MS, 6,7 and matrix-assisted laser desorption/ionization (MALDI) using quadrupole time-of-flight (Q-TOF) MS/MS 5,8 were used for identification of the P. falciparum (the human malaria parasite) proteome.…”