Phosphopeptide mapping of the wild-type and mutant hTTP proteins from transfected human cells.

<p>Wild-type and mutant hTTP proteins were labeled with [³²P]-orthophosphate in HEK293 cells. His-hTTP was purified and digested by trypsin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100977#pone-0100977-g005" target="_blank">Figure 5</a> legend. The trypsin-digested peptides were separated by reverse phase HPLC and eluted from the column at 0.5 mL/min with 20-bed volume of a linear gradient. The fractions were collected at 0.25 mL/well in 96-well plates. The radioactivity of every fraction was counted and plotted. The radioactivity in the plots from the wild-type proteins was five times of the actual radioactivity for visual comparisons of its phosphopeptides to the mutant ones because of the low recovery of phosphopeptides of the wild-type protein in HPLC fractions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100977#pone-0100977-t001" target="_blank">Table 1</a>). The radioactivity in the plots from the mutant proteins was the actual radioactivity. The phosphopeptide profiles in each pair of hTTP proteins are (A) Wild-type vs. S197A, (B) S197A vs. S(197, 228)A, (C) S(197,228)A vs. S(197,218,228)A, and (D) S(197,218,228)A vs. S(197,214,218,228)A.