Identification of GYRKPPFNGSIFamide (crustacean- SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis
Identification of GYRKPPFNGSIFamide (crustacean- SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis
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DOI：
10.1016/j.ygcen.2003.10.001
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发表时间：
2004-02-01
影响因子：
2.7
通讯作者：
Nakajima, T
中科院分区：
文献类型：
--
作者：
Yasuda, A;Yasuda-Kamatani, Y;Nakajima, T
与生物蛋白质和肽的纯化方案相关的新概念已被设计为“拓扑质谱分析”，结合使用组织切片的MALDI-TOF MS、从组织提取物的色谱纯化、用于确定前体结构的分子克隆以及用于阐明其翻译后修饰的毛细管LC-MS/MS分析。在实际应用中，我们从克氏原螯虾（Procambarus clarkii）大脑中鉴定出一种α-酰胺化神经肽。最初，通过直接MALDI-TOF MS分析发现MS数约为m/z 1382，并对大脑副叶切片进行分析。在用脑提取物进行两步反相HPLC分离后，将1381 Da肽的结构测序为GYRKPPFNGSIFamide（命名为甲壳动物-SIFamide）。随后，cDNA已被表征并编码76个氨基酸的前体蛋白，其含有信号序列、一个拷贝的GYRKPPFNGSIFG和一个额外的肽。RT-PCR分析表明，该神经肽的mRNA在螯虾的整个神经系统中均有表达。免疫组化结果显示，该神经肽分布于嗅叶、副叶、嗅球束和嗅叶细胞中。此外，数据库检索显示，在果蝇基因组文库中存在AYRKPPFNGSIFamide的同源序列，并且从灰肉蝇Neobellieria bullata分离的AYRKPPFNGSIFamide和从巨型虎虾Penaeus monodon分离的GYRKPPFNGSIFamide具有同源序列。这些结果表明，神经肽家族可能广泛分布于节肢动物体内，并在神经系统中发挥重要作用。(C)2003年爱思唯尔公司All rights reserved.
A new concept relating to the purification protocol for biological proteins and peptides has been designed as "topological mass spectrometry analysis," in combination with MALDI-TOF MS using slices of tissues, chromatographic purification from the extract of tissues, molecular cloning for the determination of the precursor structure, and capillary LC-MS/MS analysis for elucidation of its posttranslational modifications. In an actual application, we identified an alpha-amidated neuropeptide from the red swamp crayfish (Procambarus clarkii) brain. Initially, an MS number of around m/z 1382 was found by the direct MALDI-TOF MS analysis with slices of the accessory lobe of the brain. After two steps of reversed-phase HPLC separation with brain extract, the structure of a 1381 Da peptide was sequenced to the GYRKPPFNGSIFamide (named crustacean-SIFamide). Subsequently, the cDNA has been characterized and encodes a 76 amino acid precursor protein that contains a signal sequence, one copy of GYRKPPFNGSIFG and one additional peptide. The RT-PCR analysis implied that the mRNA of the neuropeptide was expressed throughout the nervous system of the crayfish. Furthermore, immunostaining demonstrated that the neuropeptide is distributed in the olfactory lobe, accessory lobe, olfactory globular tract, and olfactory lobe cells. In addition, database searches revealed that there are homologous sequences of the AYRKPPFNGSIFamide in the genome library of fruitfly Drosophila melanogaster and AYRKPPFNGSLFamide isolated from the grey fleshfly Neobellieria bullata, and GYRKPPFNGSIFamide isolated from the giant tiger prawn Penaeus monodon. These results suggested that the neuropeptide family might be widely distributed in arthropods and plays a significant role in the nervous system. (C) 2003 Elsevier Inc. All rights reserved.