MASS-SPECTROMETRIC STUDY ON THE IN-VIVO POSTTRANSLATIONAL MODIFICATION OF GAP-43

GAP-43 isolated from calf brain was analyzed by the electrospray mass spectrometry. The mass spectrum of the intact protein showed two speci es with a mass difference of SO Da, suggesting that the isolated GAP-4 3 contains phosphorylated species. To establish the in vivo phosphoryl ation sites, the protein was digested with trypsin, and analyzed by th e liquid chromatography/mass spectrometry technique, in which a capill ary reversed-phase chromatography column was connected on Line to an e lectrospray mass spectrometer. Two pairs of peptides with a mass diffe rence of 80 Da were observed. From the tandem mass spectrometry, two n ovel phosphorylation sites (Thr-87 and Ser-152) were identified. The n ovel phosphorylation sites contain proline immediately after the phosp horylated serines. No phosphorylated peptide was detected correspondin g to the protein kinase C or casein kinase II phosphorylation sites. A peptide corresponding to the acetylated N-terminal peptide was also i dentified. The mass of the peptide suggests that the 2 cysteinyl resid ues are not palmitoylated but form a disulfide bridge.