Proteome characterization of the unsequenced psychrophile Pedobacter cryoconitis using 15N metabolic labeling, tandem mass spectrometry, and a new bioinformatic workflow
Proteome characterization of the unsequenced psychrophile Pedobacter cryoconitis using 15N metabolic labeling, tandem mass spectrometry, and a new bioinformatic workflow
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使用 15N 代谢标记、串联质谱和新的生物信息学工作流程对未测序的嗜冷 Pedobacter Cryoconitis 进行蛋白质组表征
DOI：
10.1002/pmic.201100159
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发表时间：
2012
期刊：
影响因子：
3.4
通讯作者：
P. Wright
中科院分区：
文献类型：
--
作者：
Ana G. Pereira;R. Margesin;P. Wright
没有测序基因组和缺乏完整蛋白质数据库的生物体在蛋白质鉴定和定量方面遇到了更多的复杂性。从头测序、新的生物信息学工具和质谱学(MS)技术使这一领域取得了进展。在这里，利用基于15N代谢标记、2DE、MS/MS和生物信息学工具的新工作流程，展示了一种未测序的嗜冷菌--冷肠炎杆菌的蛋白质组学特征。在蛋白质组覆盖率和吞吐量方面，比较了基于氮约束(N-约束)、直系同源搜索和从头测序N-约束相似性数据库搜索的两种生物信息学管道。结果表明，不同生长温度(1℃、20℃)和不同碳源(葡萄糖、麦芽糖)对蛋白质组的影响。从葡萄糖和麦芽糖生长的细菌中分别鉴定和验证了76个69个蛋白质，其中21个和22个在不同的生长温度下有差异表达。差异表达的蛋白质参与胁迫反应和碳水化合物代谢，在20℃时比1℃时表达更高，而抗氧化剂在1℃时上调。这项研究提供了一种替代工作流程来识别、验证和量化来自蛋白质数据库中与其他物种远缘关系的未测序生物的蛋白质。此外，它还提供了对细菌对寒冷环境的适应机制的进一步了解，以及与其他嗜冷微生物的蛋白质组学比较分析。
Organisms without a sequenced genome and lacking a complete protein database encounter an added level of complexity to protein identification and quantitation. De novo sequencing, new bioinformatics tools, and mass spectrometry (MS) techniques allow for advances in this area. Here, the proteomic characterization of an unsequenced psychrophilic bacterium, Pedobacter cryoconitis, is presented employing a novel workflow based on 15N metabolic labelling, 2DE, MS/MS, and bioinformatics tools. Two bioinformatics pipelines, based on nitrogen constraint (N‐constraint), ortholog searching, and de novo peptide sequencing with N‐constraint similarity database search, are compared based on proteome coverage and throughput. Results demonstrate the effect of different growth temperatures (1°C, 20°C) and different carbon sources (glucose, maltose) on the proteome. Seventy‐six and 69 proteins were identified and validated from the glucose‐ and maltose‐grown bacterium, respectively, from which 21 and 22 were differentially expressed at different growth temperatures. Differentially expressed proteins are involved in stress response and carbohydrate metabolism, with higher expression at 20°C than at 1°C, while antioxidants were upregulated at 1°C. This study provides an alternative workflow to identify, validate, and quantify proteins from unsequenced organisms distantly related to other species in the protein database. Furthermore, it provides further understanding on bacterial adaptation mechanisms to cold environments, and a comparative proteomic analyses with other psychrophilic microorganisms.
影响因子：
6.5
作者：
Huang, CM;Foster, KW;Tang, DCC
通讯作者：
Tang, DCC
影响因子：
4.4
作者：
Wielsch, Natalie;Thomas, Henrik;Shevchenko, Andrej
通讯作者：
Shevchenko, Andrej