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

Evaluation of peptide identifications, classified as known and novel, in the 2-day data set. All peptide spectrum matches (PSMs) belonging to the respective class were compared to known false-positive decoy hits. (A) Normalized density plot of observed precursor mass deviation indicates no difference in distribution of identified known and novel PSMs. (B) Distribution of posterior error probability (PEP) shows clear distinction between decoy PSMs and known PSMs but almost overlapping distribution of known and novel hits. (C) Length of identified peptides of both classes, but not between classes, differs from decoy identifications. AA, amino acids. (D) Observed retention time shows tight correlation to predicted hydrophobicity index (HI) by SSRCalc for both classes, while retention times of known false positives only weakly correlate. In all cases, all decoy PSMs with a q value of <0.01 were plotted as a reference. See Fig. S1 and S2 for additional data sets.

Alternative splicing in the pheromone pathway-specific kinase gene mek2. (A) Graphical representation of the canonical gene structure, including observed peptides (green bars), covering three splice junctions. The mek2 gene comprises 5 exons and was identified with a total of 50 peptides, covering 75% of the sequence. (B) Graphical representation of the alternatively spliced variant. Retention of intron 4 leads to translation into an alternative protein C terminus, identified by 6 novel peptides (orange bars). (C) Label-free quantification of both MEK2 isoforms throughout six growth conditions of S. macrospora reveals downregulation of the newly identified variant (orange, SMAC_06526.3_t2) during sexual development (BMM_5d and BMM_7d). (D) Sashimi plot visualizing the RNA-Seq coverage of both splice variants in vegetative and sexual mycelium.

Peptide-level evidence for a recoding RNA editing event. (A) Parallel reaction monitoring (PRM) transitions of peptide EDDAVFFNYR originating from a putative RNA editing event in SMAC_03693, leading to the exchange of Thr to Ala at position 255. (B) PRM transitions of the genome-encoded peptide EDDTVFFNYR. (C) Peptides were monitored in fungal cells grown for 2, 3, 5, and 7 days, with the peptide originating from edited RNA only being identified in the latter two cases.

Validation of a stop-loss editing site in the transcript for the white collar 1 protein. (A) Primary amino acid sequence of the white collar 1 protein. Editing results in the conversion of a stop codon into a tryptophan codon and extends the amino acid sequence by 131 amino acids. As a consequence, the protein carries a C-terminal histone deacetylase domain (HDAC). Peptides encoded by the canonical gene are shown in red, while the blue peptide is unique to the novel C-terminal sequence. (B) Annotated MS/MS spectrum of the editing-specific peptide observed in the 7-day data set. (C) Domain structure of the white collar 1 protein, including the HDAC domain (green box) in the extended C terminus (blue box). PAS, Per-Arnt-Sim domain; GATA, GATA-type zinc finger transcription factor domain.