Proteomic Studies of the Singapore Grouper Iridovirus*S
Proteomic Studies of the Singapore Grouper Iridovirus*S
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DOI：
10.1074/mcp.m500149-mcp200
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发表时间：
2006-02
影响因子：
7
通讯作者：
Wenjun Song;Q. Lin;S. Joshi;T. Lim;C. Hew
中科院分区：
文献类型：
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作者：
Wenjun Song;Q. Lin;S. Joshi;T. Lim;C. Hew
新加坡石斑鱼虹彩病毒（SGIV）基因组由140，131个碱基对的双链环状DNA组成，具有162个预测的开放阅读框。我们早期的研究使用MALDI-TOF MS产生的肽质量指纹导致26个病毒蛋白的鉴定。本研究旨在通过两个工作流程实现SGIV病毒蛋白质组的更全面和精确的鉴定：一维凝胶电泳（1-DE）分离，然后通过MALDI-TOF/TOF MS/MS鉴定蛋白质（1-DE-MALDI工作流程）和鸟枪蛋白质组学，其中用胰蛋白酶消化整个病毒，通过纳米LC分离所得肽，并通过MALDI-TOF/TOF MS/MS分析（LC-MALDI工作流程）。共鉴定出44种病毒蛋白，其中25种为首次报道。14种蛋白质通过1-DE-MALDI工作流程唯一地鉴定，而另外10种蛋白质仅通过LC-MALDI工作流程鉴定，其中20种蛋白质通过两种方法发现。此外，还发现13个蛋白质的N端发生了乙酰化。23个蛋白质含有预测的跨膜结构域，占总蛋白质的52.3%。RT-PCR证实了所有鉴定的病毒蛋白的转录产物。本研究中通过1-DE-MALDI和LC-MALDI工作流程鉴定的大量蛋白质显著提高了SGIV蛋白质组的覆盖率。SGIV蛋白质组是目前唯一已被广泛表征的虹彩病毒蛋白质组。我们的研究结果将为SGIV和其他虹彩病毒的生物学提供进一步的见解。
The Singapore grouper iridovirus (SGIV) genome consists of a double-stranded circular DNA of 140,131 base pairs with 162 predicted open reading frames. Our earlier study using peptide mass fingerprints generated from MALDI-TOF MS led to the identification of 26 viral proteins. The present investigation aimed to achieve a more comprehensive and precise identification of the SGIV viral proteome by two workflows: one-dimensional gel electrophoresis (1-DE) separation followed by protein identification by MALDI-TOF/TOF MS/MS (1-DE-MALDI workflow) and shotgun proteomics in which the whole virus was digested by trypsin and the resulting peptides were separated by nano-LC and analyzed by MALDI-TOF/TOF MS/MS (LC-MALDI workflow). In total, 44 viral proteins were identified, 25 of which were reported for the first time. Fourteen proteins were uniquely identified by the 1-DE-MALDI workflow, whereas another 10 proteins were only identified by the LC-MALDI workflow with 20 proteins found by both approaches. Moreover 13 proteins were found to have acetylated N termini. Twenty-three proteins identified contain predicted transmembrane domains, accounting for 52.3% of the total proteins identified. RT-PCR confirmed the transcription products of all the identified viral proteins. A large number of proteins identified by both the 1-DE-MALDI and the LC-MALDI workflows from this study have significantly enhanced the coverage of the SGIV proteome. The SGIV proteome is at present the only iridoviral proteome that has been extensively characterized. Our results should provide further insights into the biology of SGIV and other iridoviruses.