Limits...
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.

Show MeSH

Related in: MedlinePlus

Detail from a 2D gel image of proteins from P. falciparum 3D7 line showing changes in pI between ring and trophozoite stages of pRBC. The Tris-insoluble 3D7 pRBC pellets of ring stage and trophozoite stage were extracted and separated on 2D electrophoresis on pH 4–7 IEF strips followed by 12.5% SDS-PAGE. The gels were stained with Coomassie blue. The indicated protein was identified as human tropomycin by MALDI-TOF mass spectrometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2696463&req=5

Figure 1: Detail from a 2D gel image of proteins from P. falciparum 3D7 line showing changes in pI between ring and trophozoite stages of pRBC. The Tris-insoluble 3D7 pRBC pellets of ring stage and trophozoite stage were extracted and separated on 2D electrophoresis on pH 4–7 IEF strips followed by 12.5% SDS-PAGE. The gels were stained with Coomassie blue. The indicated protein was identified as human tropomycin by MALDI-TOF mass spectrometry.

Mentions: As reported previously [34], using a sensitive in vivo labeling technique together with 2D electrophoresis revealed a number of changes in P. falciparum protein profiles from the pRBC of different parasite lines (Additional file 1). These changes were not limited to proteins from parasite source, but were also found in host proteins in the erythrocyte, varying during erythrocytic development of the malaria parasite e.g., PI changes of human tropomycin between ring and trophozoite stage (Figure 1). To map the phosphorylated proteins of pRBC in 2D gels, specific antibodies to phosphorylated amino acids (tyrosine and serine/threonine) were used to probe total pRBC lysates from a developmental series. As shown in Figures 2 and Figure 3, P. falciparum infection increased protein phosphorylation along with the developmental time course. There were approximately 50 protein spots that reacted positively with anti-serine/threonine antibodies and 4–5 groups of spots that extended horizontally like a family of different isoforms. The results showed that 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 major difference between ItG and C24 was the delayed appearance of a set of phosphorylated tyrosine proteins with molecular weight from 70 to 95 kDa, such that they appeared in ItG at 22 hours post-invasion and appeared in C24 at 30 hours. There were many low abundant proteins with pI changes in both strains, especially in phosphorylated serine/threonine, but it was difficult to view them in stained gels. The phosphorylation antibodies also recognized protein spots of 30, 45 and 95 kDa on non-infected erythrocytes (see Additional file 2) indicating that phospho-proteins are present in normal RBC as well (see below). The results indicated that parasite infection produced major phosphorylation changes in RBC irrespective of the stage. The majority of the proteins that reacted with anti-phosphorylated serine/threonine antibodies were identified by using MALDI-TOF and MASCOT search with phosphorylation modifications. Figure 4 indicates the proteins identified with significant scores and details of these proteins are listed in Table 1.


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)

Detail from a 2D gel image of proteins from P. falciparum 3D7 line showing changes in pI between ring and trophozoite stages of pRBC. The Tris-insoluble 3D7 pRBC pellets of ring stage and trophozoite stage were extracted and separated on 2D electrophoresis on pH 4–7 IEF strips followed by 12.5% SDS-PAGE. The gels were stained with Coomassie blue. The indicated protein was identified as human tropomycin by MALDI-TOF mass spectrometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Detail from a 2D gel image of proteins from P. falciparum 3D7 line showing changes in pI between ring and trophozoite stages of pRBC. The Tris-insoluble 3D7 pRBC pellets of ring stage and trophozoite stage were extracted and separated on 2D electrophoresis on pH 4–7 IEF strips followed by 12.5% SDS-PAGE. The gels were stained with Coomassie blue. The indicated protein was identified as human tropomycin by MALDI-TOF mass spectrometry.
Mentions: As reported previously [34], using a sensitive in vivo labeling technique together with 2D electrophoresis revealed a number of changes in P. falciparum protein profiles from the pRBC of different parasite lines (Additional file 1). These changes were not limited to proteins from parasite source, but were also found in host proteins in the erythrocyte, varying during erythrocytic development of the malaria parasite e.g., PI changes of human tropomycin between ring and trophozoite stage (Figure 1). To map the phosphorylated proteins of pRBC in 2D gels, specific antibodies to phosphorylated amino acids (tyrosine and serine/threonine) were used to probe total pRBC lysates from a developmental series. As shown in Figures 2 and Figure 3, P. falciparum infection increased protein phosphorylation along with the developmental time course. There were approximately 50 protein spots that reacted positively with anti-serine/threonine antibodies and 4–5 groups of spots that extended horizontally like a family of different isoforms. The results showed that 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 major difference between ItG and C24 was the delayed appearance of a set of phosphorylated tyrosine proteins with molecular weight from 70 to 95 kDa, such that they appeared in ItG at 22 hours post-invasion and appeared in C24 at 30 hours. There were many low abundant proteins with pI changes in both strains, especially in phosphorylated serine/threonine, but it was difficult to view them in stained gels. The phosphorylation antibodies also recognized protein spots of 30, 45 and 95 kDa on non-infected erythrocytes (see Additional file 2) indicating that phospho-proteins are present in normal RBC as well (see below). The results indicated that parasite infection produced major phosphorylation changes in RBC irrespective of the stage. The majority of the proteins that reacted with anti-phosphorylated serine/threonine antibodies were identified by using MALDI-TOF and MASCOT search with phosphorylation modifications. Figure 4 indicates the proteins identified with significant scores and details of these proteins are listed in Table 1.

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