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

Abstract

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.

Keywords: Bijective transformation; Digestive enzymes; Frameshift; RNA–DNA difference; Unbiased analyses.

Graphical abstract

Fig. 1

Sequence (A) and its systematic transformations and corresponding translations (B–F). B) A ↔ C systematic nucleotide exchange of sequence in A; C) assuming systematic codon expansion by silent mononucleotides; D) assuming systematic mononucleotide deletion after each trinucleotide (translation identical to that in C); E) assuming systematic codon expansion by silent dinucleotides; F) assuming systematic dinucleotide deletion after each trinucleotide (translation identical to that in E). RNAs and peptides corresponding to these alternative transcriptions and translations have been previously described for human mitochondria , . For swinger transformations, A ↔ C is only one among 23 possibilities, nine symmetric of type X ↔ Y, and 14 asymmetric, of type X → Y → Z → X. Systematic deletions of mono- and dinucleotides after each trinucleotide are annotated as delRNA_3–1 and delRNA_3–2. Systematic deletions can start at the 5′ extremity of a sequence, which is indicated by delRNA_3–1.0 and delRNA_3–2.0, deletion frames can be shifted by 0–2 and 0–3 nucleotides for delRNA_3–1 and delRNA_3–2, respectively, which can be indicated by corresponding indices.