Phosphopeptide mapping and phosphoamino acid analysis. STEP isoforms from striatal homogenates were phosphorylated in the absence or presence of PKA. Gel pieces containing³²P-labeled STEP₆₁ or STEP₄₆ were digested with TPCK-trypsin. The resultant phosphopeptides were separated by two-dimensional phosphopeptide mapping and visualized by autoradiography. A, C, Tryptic digest of STEP₆₁ and STEP₄₆ after phosphorylation by endogenous kinases. B, D, Tryptic digest of STEP₆₁ and STEP₄₆ after the addition of PKA. The origin of the sample is marked O. Peptides are labeled withnumbers. The positions of phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) are shown in the rightpanel of each peptide map.