Snapshot Peptidomics of the Regulated Secretory Pathway*
Snapshot Peptidomics of the Regulated Secretory Pathway*
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DOI：
10.1074/mcp.m900044-mcp200
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发表时间：
2009-03
影响因子：
7
通讯作者：
K. Sasaki;Y. Satomi;T. Takao;N. Minamino
中科院分区：
文献类型：
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作者：
K. Sasaki;Y. Satomi;T. Takao;N. Minamino
神经元和内分泌细胞具有调节分泌通路（RSP），其中前体蛋白经过激素原转化酶（PC） 1/3或2的蛋白水解，生成生物活性肽。虽然已经描述了pc介导加工的基序((R/K)Xn(R/K)，其中n = 0,2,4或6)，但实际的加工位点不能仅从氨基酸序列中预测。我们假设，通过实验确定信号肽的切割位点和加工位点，可以促进生物活性肽的发现。然而，体内和体外的肽降解，这是广泛认可的肽组学，往往阻碍加工位点的确定。为了大规模获得RSP中产生的肽的序列信息，我们对培养的内分泌细胞进行了短暂的胞外刺激（2分钟），并使用LC-MSMS分析了释放到上清中的肽。值得注意的是，400个鉴定的肽中有387个来自已知在RSP中加工的19个前体蛋白，包括9个肽激素和神经肽前体蛋白，7个颗粒样蛋白和3个加工酶（PC1/3， PC2和肽酰甘氨酸α-酰胺化单加氧酶）。总共有373个肽具有足够的信息来预测加工位点，因为它们有信号序列切割位点、PC共识位点或单基切割位点。这里确定的几个单基裂解位点以前被证明是由pc产生的。因此，我们的方法有助于预测RSP前体蛋白的加工位点，并将加快识别隐藏在前体序列中的未知生物活性肽。
Neurons and endocrine cells have the regulated secretory pathway (RSP) in which precursor proteins undergo proteolytic processing by prohormone convertase (PC) 1/3 or 2 to generate bioactive peptides. Although motifs for PC-mediated processing have been described ((R/K)Xn(R/K) where n = 0, 2, 4, or 6), actual processing sites cannot be predicted from amino acid sequences alone. We hypothesized that discovery of bioactive peptides would be facilitated by experimentally identifying signal peptide cleavage sites and processing sites. However, in vivo and in vitro peptide degradation, which is widely recognized in peptidomics, often hampers processing site determination. To obtain sequence information about peptides generated in the RSP on a large scale, we applied a brief exocytotic stimulus (2 min) to cultured endocrine cells and analyzed peptides released into supernatant using LC-MSMS. Of note, 387 of the 400 identified peptides arose from 19 precursor proteins known to be processed in the RSP, including nine peptide hormone and neuropeptide precursors, seven granin-like proteins, and three processing enzymes (PC1/3, PC2, and peptidyl-glycine α-amidating monooxygenase). In total, 373 peptides were informative enough to predict processing sites in that they have signal sequence cleavage sites, PC consensus sites, or monobasic cleavage sites. Several monobasic cleavage sites identified here were previously proved to be generated by PCs. Thus, our approach helps to predict processing sites of RSP precursor proteins and will expedite the identification of unknown bioactive peptides hidden in precursor sequences.