Amino acid substitutions may substantially alter protein stability and function1,2. However, the contribution of substitutions that arise from alternate translation (deviations from the genetic code) is unknown. Here to address this issue, we analysed deep proteomic, transcriptomic and genomic data from more than 1,000 human samples, including 6 cancer types and 26 healthy human tissues. This global analysis identified 60,803 fragmentation spectra corresponding to 8,746 unique substitutions in proteins derived from 1,767 genes, including 1,955 confidently localized sites. Some substitutions were shared across samples, whereas others exhibited strong tissue-type and cancer specificity. Notably, products of alternate translation were more abundant than their canonical counterparts for hundreds of proteins, which suggests that there is sense-codon recoding. Recoded proteins included transcription factors, proteases, signalling proteins and proteins associated with neurodegeneration. Mechanisms that contribute to substitution abundance included protein stability, codon frequency, codon–anticodon mismatches and RNA modifications. We also characterized how alternatively translated proteoform ratios vary across protein domains, tissue types and cancers. These ratios were positively associated with intrinsically disordered regions and genetic polymorphisms in the gnomAD database, although the polymorphisms could not account for the substitutions. The sequence, relative abundance and the tissue specificity of alternatively translated proteins were conserved between humans and mice. These results demonstrate the contribution of alternate translation to the diversification of mammalian proteomes and its association with protein stability, tissue-specific proteomes and disease.
