A new method for the capture of surface proteins in Plasmodium falciparum parasitized erythrocyte.
BACKGROUND
We propose a new method for the selective labeling, isolation and electrophoretic analysis of the Plasmodium falciparum protein exposed on the erythrocyte cell surface. Historically, membrane surface proteins have been isolated using a surface biotinylation followed by capture of biotin-conjugated protein via an avidin/streptavidin-coated solid support. The major drawback of the standard methods has been the labeling of internal proteins due to fast internalization of biotin.


METHODS
To solve this problem, we used a biotin label that does not permeate through the membrane. As a further precaution to avoid the purification of non surface exposed proteins, we directly challenged whole labeled cells with avidin coated beads and then solubilized them using non ionic detergents.


RESULTS
A marked enrichment of most of the RBC membrane proteins known to face the external surface of the membrane validated the specificity of the method; furthermore, only small amounts of haemoglobin and cytoskeletal proteins were detected. A wide range of P. falciparum proteins were additionally described to be exposed on the erythrocyte surface. Some of them have been previously observed and used as vaccine candidates while a number of newly described antigens have been presently identified. Those antigens require further characterization and validation with additional methods.


CONCLUSIONS
Surface proteins preparations were very reproducible and identification of proteins by mass spectrometry has been demonstrated to be feasible and effective.
Department of Genetics, Biology and Biochemistry, University of Turin, 10126 Turin, Italy. emanuela.ferru@tiscali.it
Emanuela
E
Ferru
Anntonella
A
Pantaleo
Francesco
F
Turrini
D000328:Adult; D001710:Biotin / Q000378:metabolism; D004912:Erythrocytes / Q000469:parasitology*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D010274:Parasitology / Q000379:methods*; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000472:pathogenicity; D015800:Protozoan Proteins / Q000032:analysis* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D008565:Membrane Proteins; D015800:Protozoan Proteins; D001710:Biotin