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.
Taniguchi
H
H
Division of Biomedical Polymer Science, Fujita Health University, Aichi, Japan.
Suzuki
M
M
Manenti
S
S
Titani
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019922:GAP-43 Protein; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry / Q000379:methods; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry; D014357:Trypsin
D019922:GAP-43 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D014357:Trypsin