Yoder Jennifer D, Chen Tsefang S, Gagnier Cliff R, Vemulapalli Srilakshmi, Maier Claudia S, Hruby Dennis E
Oregon State University, Department of Microbiology, 220 Nash Hall, Corvallis, OR 97331-3804, USA.
Virol J. 2006 Mar 1;3:10. doi: 10.1186/1743-422X-3-10.
Although many vaccinia virus proteins have been identified and studied in detail, only a few studies have attempted a comprehensive survey of the protein composition of the vaccinia virion. These projects have identified the major proteins of the vaccinia virion, but little has been accomplished to identify the unknown or less abundant proteins. Obtaining a detailed knowledge of the viral proteome of vaccinia virus will be important for advancing our understanding of orthopoxvirus biology, and should facilitate the development of effective antiviral drugs and formulation of vaccines.
In order to accomplish this task, purified vaccinia virions were fractionated into a soluble protein enriched fraction (membrane proteins and lateral bodies) and an insoluble protein enriched fraction (virion cores). Each of these fractions was subjected to further fractionation by either sodium dodecyl sulfate-polyacrylamide gel electophoresis, or by reverse phase high performance liquid chromatography. The soluble and insoluble fractions were also analyzed directly with no further separation. The samples were prepared for mass spectrometry analysis by digestion with trypsin. Tryptic digests were analyzed by using either a matrix assisted laser desorption ionization time of flight tandem mass spectrometer, a quadrupole ion trap mass spectrometer, or a quadrupole-time of flight mass spectrometer (the latter two instruments were equipped with electrospray ionization sources). Proteins were identified by searching uninterpreted tandem mass spectra against a vaccinia virus protein database created by our lab and a non-redundant protein database.
Sixty three vaccinia proteins were identified in the virion particle. The total number of peptides found for each protein ranged from 1 to 62, and the sequence coverage of the proteins ranged from 8.2% to 94.9%. Interestingly, two vaccinia open reading frames were confirmed as being expressed as novel proteins: E6R and L3L.
尽管已经鉴定并详细研究了许多痘苗病毒蛋白，但仅有少数研究尝试对痘苗病毒粒子的蛋白质组成进行全面调查。这些项目已鉴定出痘苗病毒粒子的主要蛋白质，但在鉴定未知或含量较少的蛋白质方面进展甚微。详细了解痘苗病毒的病毒蛋白质组对于增进我们对正痘病毒生物学的理解至关重要，并且应有助于开发有效的抗病毒药物和疫苗配方。
为完成此任务，将纯化的痘苗病毒粒子分离成富含可溶性蛋白的部分（膜蛋白和侧体）和富含不溶性蛋白的部分（病毒粒子核心）。这些部分中的每一个都通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳或反相高效液相色谱进一步分离。可溶性和不溶性部分也未经进一步分离直接进行分析。通过用胰蛋白酶消化制备用于质谱分析的样品。胰蛋白酶消化产物使用基质辅助激光解吸电离飞行时间串联质谱仪、四极杆离子阱质谱仪或四极杆 - 飞行时间质谱仪（后两种仪器配备电喷雾电离源）进行分析。通过针对我们实验室创建的痘苗病毒蛋白质数据库和非冗余蛋白质数据库搜索未解释的串联质谱来鉴定蛋白质。
在病毒粒子中鉴定出63种痘苗病毒蛋白。每种蛋白质发现的肽总数范围为1至62，蛋白质的序列覆盖率范围为8.2%至94.9%。有趣的是，两个痘苗病毒开放阅读框被确认为表达新蛋白质：E6R和L3L。
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