(A) Hydrazine and hydroxylamine cleavage of octanoylated ghrelin. Reaction mixtures were incubated 1 min at 30°C with 10 μM Ghrelin27 or 100 μM Ghrelin10, quenched in 2% SDS, bound to streptavidin resin, and washed. Capped columns were then incubated overnight with 1 mL of 1M hydrazine or hydroxylamine; 1 M MES buffer was used as a control. Beads and supernatants were then scintillation counted.
(B) Ghrelin10 (Ser3) and Dap3-Ghrelin10 were treated with hydrazine or MES as in (A).