Figure 4.

CLASPI approach to identify H3T3-Phos-binding proteins. In a “Forward” experiment, the “light” (green symbols) and “heavy” (red symbols) lysates were photo-cross-linked with probe 3 and probe C, respectively. The cross-linked proteins were then conjugated to biotin through click chemistry and isolated with streptavidin beads. After trypsin digestion, the peptide mixtures were analyzed LC-MS/MS. In a “reverse” experiment, the probes were simply switched in the photo-cross-linking reaction. Proteins that showed a high SILAC ratio (L/H) in the “forward” experiment and a low ratio (L/H) in the “reverse” experiment were likely selective H3T3-Phos-binding proteins.