Comparative SILAC proteomic analysis of Trypanosoma brucei bloodstream and procyclic lifecycle stages

Abstract

The protozoan parasite Trypanosoma brucei has a complex digenetic lifecycle between a mammalian host and an insect vector, and adaption of its proteome between lifecycle stages is essential to its survival and virulence. We have optimized a procedure for growing Trypanosoma brucei procyclic form cells in conditions suitable for stable isotope labeling by amino acids in culture (SILAC) and report a comparative proteomic analysis of cultured procyclic form and bloodstream form T. brucei cells. In total we were able to identify 3959 proteins and quantify SILAC ratios for 3553 proteins with a false discovery rate of 0.01. A large number of proteins (10.6%) are differentially regulated by more the 5-fold between lifecycle stages, including those involved in the parasite surface coat, and in mitochondrial and glycosomal energy metabolism. Our proteomic data is broadly in agreement with transcriptomic studies, but with significantly larger fold changes observed at the protein level than at the mRNA level.

Figure 1. Growth of T. brucei procyclic form cells in original SDM-79 and SILAC labelling media.

A. Cumulative growth curve. Growth in original SDM-79 containing non-dialysed FBS (open squares) is shown in parallel to SDM-79+R₀K₀ (open circles) and SDM-79+R₆K₆ (closed circles), both containing dialysed FBS. B. DIC light microscopy. T. brucei procyclic cells grown in original SDM-79, SDM-79+R₀K₀ or SDM-79+R₆K₆ for ten days were fixed in 4% paraformaldehyde and DIC images acquired on a Zeiss confocal microscope.

Figure 2. Proteomics workflow.

Procyclic cells were cultured in SDM-79+R₆K₆ then mixed 1∶1 with either unlabeled procyclic or bloodstream form cells. Sample complexity was reduced prior to LC-MS/MS analysis by either fractionation at the protein level by SDS-PAGE or at the peptide level by SCX chromatography.

Figure 3. Histograms of Log₂ fold change.

A. Procyclic form labeled with heavy isotopes (R₆K₆) mixed 1∶1 with unlabeled procyclic form (R₀K₀). B. Procyclic form labeled with heavy isotopes (R₆K₆) mixed 1∶1 with unlabeled bloodstream form (R₀K₀).

Figure 4. Agreement of comparative proteomic data with known biology.

Heatmap showing the Log₂ FC (procyclic to bloodstream) derived from comparative proteomic data (this study) and previous transcriptomic studies , , . Grey – not observed. Heatmap generated with GENEE (http://www.broadinstitute.org/cancer/software/GENE-E/).

Figure 5. Comparison of Proteomic and transcriptomic data.

Scatterplot of the Log₂ FC (procyclic to bloodstream) derived from comparative proteomic data (this study) and previous transcriptomic studies , , .

Figure 6. GO term enrichment.

A. Proteins with greater than ten-fold up- or down regulation, with enrichment P<0.01. B. Constitutively expressed proteins, with enrichment P<0.01.