(a) Dynamin I from 32Pi-labelled synaptosomes was isolated by a pull-down with GST-AmphI-SH3, digested with trypsin and the isolated phosphopeptides were separated by 2-D tryptic maps. An autoradiograph is shown. (b) The amount of radiation associated with each spot from panel a was quantified by Storm phosphorimager (n = 2, error bars indicate range). The specific phosphopeptides that accounted for each spot were identified by MALDI-TOF MS (shown above each bar in b; peptides phosphorylated on both Ser-774 + Ser-778 are in brackets). Tandem MS/MS was used to sequence the phosphorylation site in peptides A and D and B and E (not shown). Note that due to the presence of two Arg residues at each end of the phospho-box, peptides B and E have the Arg at either end (shown with a dashed line) and each presents in two forms which are chemically identical. The phosphopeptides were identified as the following dynamin I sequences with phosphorylation at Ser-774 (A-C) or Ser-774 + Ser-778 (D-F): A 774-783 + 80, m/z = 1,137.6; B 773-783 + 80 and/or 774-784 +80, m/z = 1,293.6; C 773-784 + 80, m/z = 1,449.8; D 774-783 + 160, m/z = 1,217.5; E 773-783 + 160 and/or 774-784 +160, m/z = 1,373.7; E 773-784 + 160, m/z = 1,529.6. (c) Phospho-Ser-774 predominates 2:1. The in vivo radiation distributed between Ser-774 and Ser-778 was expressed as a percent of the total 32Pi incorporated into all these phosphopeptides (63% Ser-774 and 37% Ser-778; n = 2, error bars indicating range are too small to be seen).