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A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance.

ABSTRACT: Selected reaction monitoring mass spectrometry has been combined with the use of an isotopically labelled synthetic protein, made up of proteotypic tryptic peptides selected from parasite proteins of interest. This allows, for the first time, absolute quantification of proteins from Plasmodium falciparum. This methodology is demonstrated to be of sufficient sensitivity to quantify, even within whole cell extracts, proteins of low abundance from the folate pathway as well as more abundant "housekeeping" proteins.

SUBMITTER: Southworth PM

PROVIDER: S-EPMC3219587 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance.

Southworth Paul M PM Hyde John E JE Sims Paul F G PF

Malaria journal 20111025

Selected reaction monitoring mass spectrometry has been combined with the use of an isotopically labelled synthetic protein, made up of proteotypic tryptic peptides selected from parasite proteins of interest. This allows, for the first time, absolute quantification of proteins from Plasmodium falciparum. This methodology is demonstrated to be of sufficient sensitivity to quantify, even within whole cell extracts, proteins of low abundance from the folate pathway as well as more abundant "housek ...[more]

PMID: 22027174

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Project description:Plasmodium falciparum is the main causative agent of human malaria. During the intraerythrocytic development cycle, P. falciparum morphology changes dramatically from circulating young rings to sequestered mature trophozoites and schizonts. Sequestered forms contribute to the pathophysiology of severe malaria as the infected erythrocytes obstruct the microvascular flow in deep organs and induce local inflammation. However, the sequestration mechanism limits the access to the corresponding parasitic form in clinical samples from patients infected with P. falciparum. To complement this deficiency, we aimed to evaluate the relevance of mRNA study as a proxy of protein expression in sequestered parasites. To do so, we conducted a proteotranscriptomic analysis using five independent P. falciparum laboratory strains samples. RNA sequencing was performed on circulating ring stage parasites and LC-MS/MS on the corresponding sequestered mature forms. All analyzes were performed within the same development cycle for direct individual correlation. mRNA expression level was assessed at the circulating ring stage and the corresponding protein expression level was measured after 18h-24h of maturation to reach the mature trophozoite stage. Overall, our results showed a strong transcriptome/transcriptome and proteome/proteome correlation between samples. Moreover, strong correlations of mRNA and proteins expressions levels were found between ring stage transcriptomes and mature forms proteomes. However, twice more transcripts were identified at ring stage than proteins at mature trophozoite stage. A high level of transcript expression did not guarantee the detection of the corresponding protein. Finally, we pointed out discrepancies at the individual gene level. Taken together, our results show that transcripts and proteins expression are overall correlated. However, mRNA abundance is not a perfect proxy of protein expression at the individual level. Importantly, our study shows limitations of the “blind” use of RNA-seq and the importance of multi-omics approaches for P. falciparum blood stage study in clinical samples.

Dataset Information

A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance.

Publications

A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance.

Similar Datasets

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