MALDI-TOF/TOF de novo sequence analysis of 2-D PAGE-separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome
MALDI-TOF/TOF de novo sequence analysis of 2-D PAGE-separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome
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DOI：
10.1002/elps.200500959
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发表时间：
2006-07-01
期刊：
影响因子：
2.9
通讯作者：
Van Beeumen, Jozef
中科院分区：
文献类型：
--
作者：
Samyn, Bart;Sergeant, Kjell;Van Beeumen, Jozef
由于蛋白质鉴定依赖于与序列数据库的匹配，因此高通量蛋白质组学目前主要限于可获得全面序列数据库的那些物种。对基因组未测序生物的蛋白质的鉴定主要依赖于同源性搜索。在这里，我们报告使用一个简化的，基于凝胶的，化学衍生化的从头序列分析策略，使用MALDI-TOF/TOF质谱仪。该方法允许测定长度高达20个氨基酸残基的从头肽序列。该协议被应用于蛋白质组学研究的2-D PAGE分离的蛋白质从嗜盐螺旋体，极端嗜真细菌，尚未测序的基因组。使用三种不同的基于同源性的搜索算法，我们能够使用亚皮摩尔数量的蛋白质从这种生物体中识别出30多种蛋白质。
Because protein identifications rely on matches with sequence databases, high-throughput proteomics is currently largely restricted to those species for which comprehensive sequence databases are available. The identification of proteins derived from organisms with unsequenced genomes mainly depends on homology searching. Here, we report the use of a simplified, gel-based, chemical derivatization strategy for de novo sequence analysis using a MALDI-TOF/TOF mass spectrometer. This approach allows the determination of de novo peptide sequences of up to 20 amino acid residues in length. The protocol was applied on a proteomic study of 2-D PAGE-separated proteins from Halorhodospira halophila, an extremophilic eubacterium with yet unsequenced genome. Using three different homology-based search algorithms, we were able to identify more than 30 proteins from this organism using subpicomole quantities of protein.