ABSTRACT: Proteogenomics, the combination of proteomics, genomics and transcriptomics, has considerably improved genome annotation in under-studied phylogenetic groups, where homology information is missing. Yet, it can also be advantageous when re-investigating well-annotated genomes. Here, we apply 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 detection of 104 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. Co-expression and phylostratigraphic analysis of the refined proteome suggests new functions in evolutionary young genes correlated with distinct developmental stages. In conclusion, our advanced proteogenomics approach is highly supportive to promote functional studies of model systems. Proteogenomics, the combination of proteomics, genomics and transcriptomics, has considerably improved genome annotation in under-studied phylogenetic groups, where homology information is missing. Yet, it can also be advantageous when re-investigating well-annotated genomes. Here, we apply 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 detection of 104 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. Co-expression and phylostratigraphic analysis of the refined proteome suggests new functions in evolutionary young genes correlated with distinct developmental stages. In conclusion, our advanced proteogenomics approach is highly supportive to promote functional studies of model systems.
Bernhard Blank-Landeshammer 
