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Identification of phosphorylated proteins in erythrocytes infected by the human malaria parasite Plasmodium falciparum.

Wu Y, Nelson MM, Quaile A, Xia D, Wastling JM, Craig A - Malar. J. (2009)

Bottom Line: Protein phosphorylation is a major process in P. falciparum-parasitized erythrocytes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. ywu@liverpool.ac.uk

ABSTRACT

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.

Conclusion: 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.

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1D gel image of proteins eluted from a phospho-protein purification column. Lane 1, proteins with mock treatment, lane 2 proteins treated with SAP prior to purification/enrichment for phospho-proteins. The arrows indicate the cutting side and the numbers represent the bands excised and processed for LC/MS/MS analysis.
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Figure 5: 1D gel image of proteins eluted from a phospho-protein purification column. Lane 1, proteins with mock treatment, lane 2 proteins treated with SAP prior to purification/enrichment for phospho-proteins. The arrows indicate the cutting side and the numbers represent the bands excised and processed for LC/MS/MS analysis.

Mentions: To improve the sensitivity of detection, phosphorylated proteins purified/enriched by affinity chromatography techniques were separated by 1D SDS-PAGE and identified by nano-flow LC/MS/MS. Figure 5 shows the gel images of the proteins treated with either SAP or mock buffer and then subjected to a PhosProtein Purification column (Qiagen). The phosphorylated proteins were eluted after extensive washing, precipitated and separated in 1-DE. The arrows indicate bands that were excised for LC/MS/MS analysis. When the whole pRBC lysate was pre-treated with phosphatase, the level and extent of proteins seen on the gel (lane 2) and therefore available for affinity purification was greatly reduced (NB: equal amounts of proteins were loaded on the affinity column). 34 bands were excised from lane 1 and the proteins identified represent a shotgun phosphoproteome of ItG trophozoites. All the peptide ions generated from LC/MS/MS were searched against NCBI databases using MASCOT search with and without phosphorylation modifications.


Identification of phosphorylated proteins in erythrocytes infected by the human malaria parasite Plasmodium falciparum.

Wu Y, Nelson MM, Quaile A, Xia D, Wastling JM, Craig A - Malar. J. (2009)

1D gel image of proteins eluted from a phospho-protein purification column. Lane 1, proteins with mock treatment, lane 2 proteins treated with SAP prior to purification/enrichment for phospho-proteins. The arrows indicate the cutting side and the numbers represent the bands excised and processed for LC/MS/MS analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2696463&req=5

Figure 5: 1D gel image of proteins eluted from a phospho-protein purification column. Lane 1, proteins with mock treatment, lane 2 proteins treated with SAP prior to purification/enrichment for phospho-proteins. The arrows indicate the cutting side and the numbers represent the bands excised and processed for LC/MS/MS analysis.
Mentions: To improve the sensitivity of detection, phosphorylated proteins purified/enriched by affinity chromatography techniques were separated by 1D SDS-PAGE and identified by nano-flow LC/MS/MS. Figure 5 shows the gel images of the proteins treated with either SAP or mock buffer and then subjected to a PhosProtein Purification column (Qiagen). The phosphorylated proteins were eluted after extensive washing, precipitated and separated in 1-DE. The arrows indicate bands that were excised for LC/MS/MS analysis. When the whole pRBC lysate was pre-treated with phosphatase, the level and extent of proteins seen on the gel (lane 2) and therefore available for affinity purification was greatly reduced (NB: equal amounts of proteins were loaded on the affinity column). 34 bands were excised from lane 1 and the proteins identified represent a shotgun phosphoproteome of ItG trophozoites. All the peptide ions generated from LC/MS/MS were searched against NCBI databases using MASCOT search with and without phosphorylation modifications.

Bottom Line: Protein phosphorylation is a major process in P. falciparum-parasitized erythrocytes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. ywu@liverpool.ac.uk

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus