Fig. 1.

Overview of enrichment methods for thiophosphorylated peptides and proteins. The orange box displays the work flow to prepare the peptides and protein mixtures before the enrichment. (A) Schematic of different methods to enrich thiophosphopeptides, which include titanium dioxide (TiO₂) beads, UltraLink iodoacetyl resin (iodoacetyl-beads), and an anti-thiophosphate ester antibody. After all the steps, peptides are eluted and analyzed by nanoLC-MS/MS. (B) Schematic of different methods used for thiophosphoprotein enrichment. The methods include UltraLink iodoacetyl resin (iodoacetyl-beads) and anti-thiophosphate ester antibody. After all the steps, peptides are eluted and analyzed by nanoLC-MS/MS. (C) PNBM derivatized thiophosphorylated proteins detected by Western blotting using the anti-thiophosphate ester antibody. The first lane shows that HeLa nuclei only using alkylation with PNBM (but without ATP-γ-S), and the second lane shows HeLa nuclei incubated with ATP-γ-S followed by alkylation with PNBM.