Identification of phosphorylated proteins in erythrocytes infected by the human malaria parasite Plasmodium falciparum.
BACKGROUND
Previous comparative proteomic analysis on Plasmodium falciparum isolates of different adhesion properties suggested that protein phosphorylation varies between isolates with different cytoadherence properties. But the extent and dynamic changes in phosphorylation have not been systematically studied. As a baseline for these future studies, this paper examined changes in the phosphoproteome of parasitized red blood cells (pRBC).


METHODS
Metabolic labelling with [35S] methionine on pRBC and 2D gel electrophoresis (2-DE) has previously been used to show the expression of parasite proteins and changes in protein iso-electric point (PI). 2-DE of different parasite strains was combined with immunoblotting using monoclonal antibodies specifically to phosphorylated serine/threonine and tyrosine, to obtain the phosphorylation profiles throughout the erythrocytic lifecycle. Affinity chromatography was used to purify/enrich phosphorylated proteins and these proteins from mature trophozoite stages which were identified using high-accuracy mass spectrometry and MASCOT search.


RESULTS
2D-immunoblots showed that P. falciparum infection greatly increased phosphorylation of a set of proteins in pRBC, the dominant size classes for phosphorylated tyrosine proteins were 95, 60, 50 and 30 kDa and for phosphorylated serine/threonine were 120, 95, 60, 50, 43, 40 and 30 kDa. The most abundant molecules from 2D-gel mapping of phosphorylated proteins in ItG infected RBCs were identified by MALDI-TOF. A proteomic overview of phosphorylated proteins in pRBC was achieved by using complementary phosphorylated protein enrichment techniques combined with nano-flow LC/MS/MS analysis and MASCOT MS/MS ions search with phosphorylation as variable modifications. The definite phosphoproteins of pRBC are reported and discussed.


CONCLUSIONS
Protein phosphorylation is a major process in P. falciparum-parasitized erythrocytes. Preliminary screens identified 170 P. falciparum proteins and 77 human proteins as phosphorylated protein in pRBC, while only 48 human proteins were identified in the corresponding fractions from uninfected RBC. Refinement of the search to include significant ion scores indicating a specific phospho-peptide identified 21 P. falciparum proteins and 14 human proteins from pRBC, 13 host proteins were identified from normal RBC. The results achieved by complementary techniques consistently reflect a reliable proteomic overview of pRBC.
Wu
Yang
Y
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. ywu@liverpool.ac.uk
Nelson
Morag M
MM
Quaile
Andrew
A
Xia
Dong
D
Wastling
Jonathan M
JM
Craig
Alister
A
D000818:Animals; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004912:Erythrocytes / Q000737:chemistry* / Q000469:parasitology*; D006801:Humans; D013058:Mass Spectrometry; D008970:Molecular Weight; D010766:Phosphorylation; D010963:Plasmodium falciparum / Q000254:growth & development*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D015800:Protozoan Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D011506:Proteins; D020543:Proteome; D015800:Protozoan Proteins