Combination of Proteogenomics with Peptide De Novo Sequencing Identifies New Genes and Hidden Posttranscriptional Modifications
Combination of Proteogenomics with Peptide De Novo Sequencing Identifies New Genes and Hidden Posttranscriptional Modifications
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DOI：
10.1128/mbio.02367-19
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发表时间：
2019-09-01
期刊：
影响因子：
6.4
通讯作者：
Sickmann, A.
中科院分区：
文献类型：
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作者：
Blank-Landeshammer, B.;Teichert, I.;Sickmann, A.
蛋白质组学结合了蛋白质组学、基因组学和转录组学，并在缺乏同源性信息的研究较少的系统发育类群中极大地改善了基因组注释。此外，当重新研究注释良好的基因组时，它可能是有利的。在这里，我们应用了一种先进的蛋白质组学方法，将标准蛋白质组学与肽从头测序相结合，对研究得很好的模式真菌大孢子菌进行了完善的注释。我们调查了来自不同发育和生理条件的样本，结果检测到104个迄今隐藏的蛋白质和575个基因的注释变化，其中包括389个剪接位点的精化。值得注意的是，我们的方法提供了来自A-to-I RNA编辑的113个单一氨基酸变异和15个C-末端蛋白质延伸的肽水平证据，这是最近在真菌中发现的现象。对精制蛋白质组的共表达和系统地层学分析表明，在进化年轻的基因中，新功能与不同的发育阶段相关。总之，我们先进的蛋白质组学方法支持并促进了真菌模型系统的功能研究。新一代测序技术的引入大大增加了真核细胞基因组全序列的数量。这些基因组大多是自动注释的，从头算基因预测通常与基于同源性的搜索方法相结合，并经常得到转录组数据的支持。后者尤其提高了对内含子剪接位点和非翻译区的预测。然而，对翻译起始点(TIS)、选择性剪接连接和蛋白质编码潜力的正确预测仍然具有挑战性。在这里，我们提出了一种先进的蛋白质组学方法，即结合蛋白质组学和从头测序分析，结合Blast2GO和系统地层学。以模式真菌大孢子菌为例，我们提供了一个全面的蛋白质组图，不仅增加了对这种多细胞生物体在不同发育阶段的功能的了解，而且极大地提高了基因组注释的质量。
Proteogenomics combines proteomics, genomics, and transcriptomics and has considerably improved genome annotation in poorly investigated phylogenetic groups for which homology information is lacking. Furthermore, it can be advantageous when reinvestigating well-annotated genomes. Here, we applied an advanced proteogenomics approach, combining standard proteogenomics with peptide de novo sequencing, to refine annotation of the well-studied model fungus Sordaria macrospora. We investigated samples from different developmental and physiological conditions, resulting in the detection of 104 so-far hidden proteins and annotation changes in 575 genes, including 389 splice site refinements. Significantly, our approach provides peptide-level evidence for 113 single-amino-acid variations and 15 C-terminal protein elongations originating from A-to-I RNA editing, a phenomenon recently detected in fungi. Coexpression and phylostratigraphic analysis of the refined proteome suggest that new functions in evolutionarily young genes correlate with distinct developmental stages. In conclusion, our advanced proteogenomics approach supports and promotes functional studies of fungal model systems.IMPORTANCE Next-generation sequencing techniques have considerably increased the number of completely sequenced eukaryotic genomes. These genomes are mostly automatically annotated, and ab initio gene prediction is commonly combined with homology-based search approaches and often supported by transcriptomic data. The latter in particular improve the prediction of intron splice sites and untranslated regions. However, correct prediction of translation initiation sites (TIS), alternative splice junctions, and protein-coding potential remains challenging. Here, we present an advanced proteogenomics approach, namely, the combination of proteogenomics and de novo peptide sequencing analysis, in conjunction with Blast2GO and phylostratigraphy. Using the model fungus Sordaria macrospora as an example, we provide a comprehensive view of the proteome that not only increases the functional understanding of this multicellular organism at different developmental stages but also immensely enhances the genome annotation quality.