title: 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.
authors.affiliation: Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, 0002, South Africa.
authors.forename: Salome
authors.initials: S
authors.lastname: Smit
authors.forename: Stoyan
authors.initials: S
authors.lastname: Stoychev
authors.forename: Abraham I
authors.initials: AI
authors.lastname: Louw
authors.forename: Lyn-Marie
authors.initials: LM
authors.lastname: Birkholtz
mesh_terms: D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004912:Erythrocytes; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009942:Organometallic Compounds / Q000737:chemistry; D010963:Plasmodium falciparum / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D012394:Rosaniline Dyes / Q000737:chemistry; D016622:Silver Staining; D053058:Trophozoites / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D020033:Protein Isoforms; D020543:Proteome; D015800:Protozoan Proteins; D012394:Rosaniline Dyes; C515601:Sypro Ruby; C048139:Coomassie blue