Taniguchi H, Suzuki M, Manenti S, Titani K
Division of Biomedical Polymer Science, Fujita Health University, Aichi, Japan.
J Biol Chem. 1994 Sep 9;269(36):22481-4.
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.
采用电喷雾质谱法对从小牛脑中分离出的GAP - 43进行了分析。完整蛋白质的质谱图显示出两种质量相差80道尔顿的物质，这表明分离出的GAP - 43含有磷酸化形式。为确定体内磷酸化位点，用胰蛋白酶消化该蛋白质，并采用液相色谱/质谱技术进行分析，其中毛细管反相色谱柱与电喷雾质谱仪在线连接。观察到两对质量相差80道尔顿的肽段。通过串联质谱法，鉴定出两个新的磷酸化位点（苏氨酸 - 87和丝氨酸 - 152）。新的磷酸化位点在磷酸化丝氨酸之后紧接着脯氨酸。未检测到与蛋白激酶C或酪蛋白激酶II磷酸化位点相对应的磷酸化肽段。还鉴定出了一个与乙酰化N末端肽相对应的肽段。该肽段的质量表明，2个半胱氨酰残基未被棕榈酰化，而是形成了二硫键。
