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Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites.

ABSTRACT: BACKGROUND: Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the correlations between abundance of transcripts and their cognate proteins remain poorly characterized. RESULTS: Here, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34 to 46 hours after invasion) based on two-dimensional differential gel electrophoresis of protein samples labeled with fluorescent dyes. For this purpose we analyzed parasite samples taken at 4-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins--including actin-I, enolase, eukaryotic initiation factor (eIF)4A, eIF5A, and several heat shock proteins--are represented by more than one isoform, presumably caused by post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal evidence of significant post-transcriptional gene expression regulation. CONCLUSIONS: Together, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra-erythrocytic development of P. falciparum.

SUBMITTER: Foth BJ

PROVIDER: S-EPMC2646281 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites.

Foth Bernardo J BJ Zhang Neng N Mok Sachel S Preiser Peter R PR Bozdech Zbynek Z

Genome biology 20081217 12

<h4>Background</h4>Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the correlations between abundance of transcri ...[more]

PMID: 19091060

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Project description:Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly.

Dataset Information

Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites.

Publications

Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites.

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