Global Quantitative SILAC Phosphoproteomics Reveals Differential Phosphorylation Is Widespread between the Procyclic and Bloodstream Form Lifecycle Stages of Trypanosoma brucei

We report a global quantitative phosphoproteomic study of bloodstream and procyclic form Trypanosoma brucei using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO2 led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model based gene enrichment analysis identified over representation of Gene Ontology terms relating to the flagella, protein kinase activity, and the regulation of gene expression. The quantitative data reveal that differential protein phosphorylation is widespread between bloodstream and procyclic form trypanosomes, with significant intraprotein differential phosphorylation. Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were identified, and these were 3-4 fold over represented among site changing >10-fold between the two lifecycle stages. A significant proportion of the T. brucei kinome was phosphorylated, with evidence that MAPK pathways are functional in both lifecycle stages. Regulation of gene expression in T. brucei is exclusively post-transcriptional, and the extensive phosphorylation of RNA binding proteins observed may be relevant to the control of mRNA stability in this organism.