Fig. 1.

Experimental setup. Tissue-culture-derived trypomastigotes were harvested and washed before incubation in DMEM at pH 5.0 for 0 h, 0.5 h, 2 h, and 9 h. After incubation, the parasites were lysed in 8 m urea. Following protein digestion, peptides were labeled with iTRAQ and combined in 1:1:1:1 ratio. Phosphopeptides were enriched via TiO₂ chromatography, and non-modified peptides (flow-through and washes) were pre-fractionated offline on a TSKGel Amide 80 HILIC HPLC column and subjected to LC-MS/MS analysis.