| 孙俊毅1，孙媛媛2，苟建重1，李 昂3 “鸟枪”蛋白组学技术鉴定牙龈卟啉单胞菌47A-1亚型外膜蛋白 |
| 论文编辑部-新丝路理论网 2011-03-31 15:29:10 作者:中华医学之家：http://www.xinxi85.com 来源: 文字大小:[大][中][小] |
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Title:
Identification of outer membrane proteins of Porphyromonas gingivalis 47A-1 by Shotgun proteomics method
- 文章编号:
- 1671-8259(2011)02-0170-05
- 作者:
- 孙俊毅1; 孙媛媛2; 苟建重1; 李 昂3
- 1. 西安交通大学医学院附属口腔医院牙周科，陕西西安 710004；2. 烟台市口腔医院牙周科，山东烟台 264001；3. 西安交通大学医学院附属口腔医院口腔医学研究中心，陕西西安 710004
- Author(s):
- SUN Jun-yi1; SUN Yuan-yuan2; GOU Jian-chong1; LI Ang3
- 1. Department of Periodontology, Stomatological Hospital, Medical School of Xi’an Jiaotong University, Xi’an 710004; 2. Department of of Periodontolgy,
Yantai Stomatological Hospital, Yantai 264001, 3. Research Center of Stomatology, Stomatological Hospital, Medical School of Xi’an Jiaotong University, Xi’an, 710004, China
- 关键词:
- ; 牙龈卟啉单胞菌; 外膜蛋白; 鸟枪法; 蛋白质组学
- Keywords:
- ; Porphyromonas gingivalis; outer membrane protein; shotgun method; proteomics
- 分类号:
- R34;R37
- DOI:
- -
- 文献标识码:
- A
- 摘要:
- 摘要：目的 利用shotgun(鸟枪)蛋白质组学策略鉴定牙龈卟啉单胞菌(Porphyromonas gingivalis, P.g)亚型47A-1外膜蛋白，深化P.g外膜蛋白的蛋白质组学研究，为进一步研究P.g外膜蛋白的致病性奠定基础。方法 提取P.g47A-1外膜蛋白，SDS-PAGE电泳分离蛋白，胶内酶解为多肽混合物，应用毛细管高效液相色谱加串联质谱分析，SEQUEST程序进行数据库搜索并鉴定蛋白。结果 用考马斯亮蓝法染色凝胶分离的蛋白，发现P.g47A-1蛋白条带主要在14.3～97ku之间。酶解的肽段经毛细管高效液相色谱分离，得到肽段水平的总离子流图，分析肽段序列后发现P.g47A-1蛋白有623个唯一肽段，鉴定出136种蛋白，包括了51种已知蛋白，85种未知蛋白。结论 Shotgun技术可分析出P.g47A-1大量未知蛋白，尤其是丰度低、疏水性强的外膜蛋白，优于二维凝胶电泳(two dimensional gel electrophoresis, 2-DE)等传统蛋白质组学方法，为进一步研究P.g外膜蛋白的致病性奠定了基础。
- Abstract:
- ABSTRACT: Objective To identify outer membrane proteins of Porphyromonas gingivalis (P.g.) by shotgun proteomics method so as to lay a foundation for further study of pathogenicity and proteomics of P.g. Methods P.g47A-1 were cultured and collected when they were in good conditions. Outer membrane proteins of Pg 47A-1 were extracted by differential centrifugation and separated by SDS-PAGE. We selected trypsin for protein hydrolysis in SDS-PAGE gel, separated the peptide fragments using cap HPLC, and analyzed each peptide fragment in MS/MS. With the help of Sequest software, the NCBI database was searched for the identification of outer membrane proteins of P.g47A-1. Results The proteins in gels were stained using Coomassie Brilliant Blue, and the outer membrane protein bands were mainly located between 14.4 and 97ku. Separation of the peptides was achieved by capillary high performance liquid chromatography and identified using the total ion flow diagram. Peptide fragment sequence showed that a total of 623 peptides, 136 differential proteins were identified, of which 51 proteins were known and the other 85 proteins were unknown. Conclusion Shotgun technique can analyze a large number of unknown proteins in P.g47A-1, especially outer membrane proteins with low abundance and strong hydrophobicity. Compared with two-dimensional gel electrophoresis, shotgun proteomics might be a better method for the analysis of outer membrane proteins. The findings on OMPS of P.g in the present study might supply some clues for further understanding of its pathogenic mechanisms.
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基金资助：国家自然科学基金资助项目(No.30500560)
Supported by the National Natural Science Foundation of China (No.30500560)
通讯作者：李昂，副研究员. E-mail: drliang@mail.xjtu.edu.cn
作者简介：孙俊毅(1979-)，男(汉族)，住院医师. 研究方向：牙周病的病因及预防. E-mail: sjy0224@mail.xjtu.edu.cn
中华医学之家：http://www.xinxi85.com
投稿信箱：zhyxzj85@163.com
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