Fig. 3. Analysis of peptides from eIF2Bε phosphorylated by GSK3. eIF2Bε made in insect cells was radiolabelled in vitro using GSK3. (A) This was then subjected to digestion by Asp-N and the resulting peptides were analysed by reverse-phase (C₁₈) HPLC. The dotted line indicates the percentage (v/v) of CH₃CN. (B and C) The material in fractions 65 (B) or 74 (C) from the HPLC was analysed by two-dimensional mapping. (D and E) These fractions (as indicated) were subjected to solid-phase sequencing, and release of radioactivity was determined at each cycle. The sequences of the peptides are also displayed (as shown by the release of radioactivity and the masses of the peptides determined by mass spectrometry, see text: Sp denotes phosphoserine). (F and G) Two-dimensional analysis of tryptic peptides from either bacterially expressed rat eIF2Bε phosphorylated in vitro by DYRK2 (F) or rat eIF2Bε expressed in insect cells and phosphorylated by GSK3 (G). Peptides t1 and t3 (see text) are indicated.