A 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 species with a mass difference of 80 Da, suggesting that the isolated GAP-43 contains phosphorylated species. To establish the in vivo phosphorylation sites, the protein was digested with trypsin, and analyzed by the liquid chromatography/mass spectrometry technique, in which a capillary reversed-phase chromatography column was connected on line to an electrospray mass spectrometer. Two pairs of peptides with a mass difference of 80 Da were observed. From the tandem mass spectrometry, two novel phosphorylation sites (Thr-87 and Ser-152) were identified. The novel phosphorylation sites contain proline immediately after the phosphorylated serines. No phosphorylated peptide was detected corresponding to the protein kinase C or casein kinase II phosphorylation sites. A peptide corresponding to the acetylated N-terminal peptide was also identified. The mass of the peptide suggests that the 2 cysteinyl residues are not palmitoylated but form a disulfide bridge.