Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species:: Comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes
Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species:: Comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes
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DOI：
10.1016/j.jbiotec.2008.02.007
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发表时间：
2008-08-31
影响因子：
4.1
通讯作者：
Weber, Andreas P. M.
中科院分区：
文献类型：
--
作者：
Braeutigam, Andrea;Shrestha, Roshan P.;Weber, Andreas P. M.
蛋白质组学是建立和比较特定组织、细胞类型或亚细胞结构的蛋白质含量的有价值的工具。它在非模式物种中的使用目前受到限制，因为肽的鉴定严重依赖于序列数据库。在这项研究中，我们探讨了一个初步的cDNA数据库的非模式物种豌豆创建了少量的大规模并行焦磷酸测序（MPSS）运行其用于蛋白质组学的潜力，并比较它的综合cDNA数据库从蒺藜苜蓿和拟南芥创建的桑格测序。每个数据库用于鉴定来自豌豆叶叶绿体包膜制备物的蛋白质。结果表明，豌豆数据库确定了更多的蛋白质与更高的准确性，虽然序列质量低，序列重叠群短的数据库相比，从模式物种。虽然通过降低成功蛋白质鉴定的阈值可以潜在地增加非物种特异性数据库中鉴定的蛋白质的数量，但这种策略显著增加了错误鉴定的蛋白质的数量。非物种特异性数据库的鉴定率与光谱丰度相关，但与预测的膜螺旋含量无关，强保守性是必要的，但不足以用于非物种特异性数据库的蛋白质鉴定。可以得出结论，cDNA的大规模平行测序大大增加了非模式物种中蛋白质组学的能力。(C)2008 Elsevier B.V.保留所有权利。
Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides Critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify Proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for Successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and Strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively Parallel sequencing of cDNAs substantially increases the power Of proteomics in non-model species. (C) 2008 Elsevier B.V. All rights reserved.