Combination of Proteogenomics with Peptide De Novo Sequencing Identifies New Genes and Hidden Posttranscriptional Modifications

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.