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.