Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite

Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite

Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite

Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite

Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite

Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite

Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite

Proteomic profiling of P750-clpP1P2DAS in the presence and absence of ATc reveals a wide array of potential Clp protease substrates.

<p>(A) In triplicate, P750-clpP1P2DAS was grown for 48 hours in the absence, denoted “wt”, or presence of ATc (1.5 µg/mL), denoted “mut”, from a starting OD₆₀₀ of 0.02. Immunoblotting of protein lysates with α-ClpP2 and α-RpoB (loading control) demonstrates degree of ClpP2-DAS depletion in mut cells. Samples were then used for TMT₆ MS3-based quantitative proteomics. The specific TMT reagent used for each condition is listed under the immunoblot. (B) Normalized, summed intensities for all quantified proteins was used to perform Pearson correlational hierarchical clustering of biological replicates. (C) The Log2 ratio of average mutant protein intensity to average wildtype protein intensity plotted against the p-value determined by t-test, grouping the three biological replicates. The threshold for over-representation was set at an average ratio of greater than equal to 2, while the cut-off for under-representation was 0.5. In both instances, p-values below 0.01 were deemed significant. Proteins considered for further analysis are denoted in red. (D) The relative quantity of specific proteins plotted across the six TMT channels, for highly (left) and moderately (center) over-represented, and under-represented (right) proteins in the mutant versus wildtype conditions.</p

Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite

Global Analysis of Protein Expression and Phosphorylation of Three Stages of <i>Plasmodium falciparum</i> Intraerythrocytic Development

During asexual intraerythrocytic development, <i>Plasmodium falciparum</i> diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the <i>Plasmodium</i> asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the <i>Plasmodium</i> proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase–substrate pairs operative in various signaling networks in the parasite