Unbiased Mitoproteome Analyses Confirm Non-canonical RNA, Expanded Codon Translations.

Proteomic MS/MS mass spectrometry detections are usually biased towards peptides cleaved by experimentally added digestion enzyme(s). Hence peptides resulting from spontaneous degradation and natural proteolysis usually remain undetected. Previous analyses of tryptic human proteome data (cleavage after K, R) detected non-canonical tryptic peptides translated according to tetra- and pentacodons (codons expanded by silent mono- and dinucleotides), and from transcripts systematically (a) deleting mono-, dinucleotides after trinucleotides (delRNAs), (b) exchanging nucleotides according to 23 bijective transformations. Nine symmetric and fourteen asymmetric nucleotide exchanges (X ↔ Y, e.g. A ↔ C; and X → Y → Z → X, e.g. A → C → G → A) produce swinger RNAs. Here unbiased reanalyses of these proteomic data detect preferentially non-canonical tryptic peptides despite assuming random cleavage. Unbiased analyses couldn't reconstruct experimental tryptic digestion if most detected non-canonical peptides were false positives. Detected non-tryptic non-canonical peptides map preferentially on corresponding, previously described non-canonical transcripts, as for tryptic non-canonical peptides. Hence unbiased analyses independently confirm previous trypsin-biased analyses that showed translations of del- and swinger RNA and expanded codons. Accounting for natural proteolysis completes trypsin-biased mitopeptidome analyses, independently confirms non-canonical transcriptions and translations.