Identification of Plasmodium falciparum nuclear proteins by mass spectrometry and proposed protein annotation

Abstract

The nuclear proteome of Plasmodium falciparum results from the continual shuttle of proteins between the cell cytoplasm-nucleus and vice versa. Using shotgun proteomics tools, we explored the nuclear proteins of mixed populations of Plasmodium falciparum extracted from infected erythrocytes. We combined GeLC-MS/MS and 2D-LC-MS/MS with a peptide ion exclusion procedure in order to increase the detection of low abundant proteins such as those involved in gene expression. We have identified 446 nuclear proteins covering all expected nuclear protein families involved in gene regulation. All structural ribosomal (40S and 60S) proteins were identified which is consistent with the nuclear localization of ribosomal biogenesis. Proteins involved in the translation machinery were also found suggesting that translational events might occur in the nucleus in P. falciparum as previously hypothesized in eukaryotes. These data were compared to the protein list established by PlasmoDB and submitted to Plasmobase a recently reported Plasmodium annotation website to propose new functional putative annotation of several unknown proteins found in the nuclear extracts.

Fig 1. Comparison between two nuclear and cytoplasmic extracts.

A. SDS-PAGE of 10 μg of two nuclear biological replicates NE1 and NE2 as well as their corresponding cytoplasmic extracts CE1 and CE2 were stained by Coomassie Blue (left). For the Western blotting experiment (right) the membrane was probed by an anti-aldolase and an anti-PfHMGB2 revealing the corresponding proteins; orange arrow for aldolase and green arrow for PfHMGB2. HMGB corresponds to 100 ng of recombinant PfHMGB2 used as a control. B. Venn diagram of the proteins identified in CE and NE extracts (the list of proteins is reported in S2 Fig).

Fig 2. Progression of the number of identified proteins by precursor ion exclusion.

Venn diagram of three technical repeats of one μg of NE2 analysed by 2D-LC-MS/MS either without exclusion (WE) or after precursor ion exclusion F.PIE and C.PIE as described in Methods and S1 Fig.

Fig 3. Venn diagram of all proteins identified by GeLC-MS/MS and 2D-LC-MS/MS.

Venn diagram of several biological and technical nuclear extract replicates analysed either by GeLC-MS/MS or 2D-LC-MS/MS in order to generate a complete list of 446 identified proteins presented in S4B Fig.

Fig 4. Gene ontology analysis.

GO term analysis was performed using PANTHER functional classification. A. Organelle GO terms distribution for CE and NE (percent of the total number of hits). B. Protein Class GO terms illustrating the relative proportion of protein molecular functions in CE and NE (percent of the total number of hits). C. Biological processes significantly overrepresented in NE compared to the complete P. falciparum gene database (Fischer test, p value < 0.01, FDR < 5%).