Figure 5.

Schematic representation of the analysis of the commitment proteome and phosphoproteome. Proteomic workflow. Ex vivo stumpy cells were incubated at 37°C in the presence or absence of CA for 1 and 3 h. An aliquot of each sample was withdrawn, washed, and cultured in the absence of CA before analysis of EP procyclin expression and reentry into the cell cycle to confirm that commitment occurred on the expected timescale. The remainder of the each sample was mixed with an equal number of Arg6Lys4 heavy isotope–labeled cultured T. brucei cells, digested with trypsin, and peptides were subjected to proteomic or phosphoproteomic analysis. Experiments were conducted in biological triplicate. SCX, strong cation exchange.