Proteogenomic Approach to UTR Peptide Identification

Recent sequencing technologies have highlighted translation
of
untranslated regions (UTRs) in genomes, although it remains unknown
whether the translated products persist in a cell. Here, we propose
a proteogenomic approach to UTR identification at the proteome level,
which has been challenging due to the lack of corresponding sequences
required for peptide spectrum matching. We address the challenge with
constructing translated UTR (tUTR) database, consisting of all hypothetical
sequences that can be translated from UTR by assuming non-AUG initiation
at near-cognate start codons and stop codon readthrough. In the analysis
of the H1299 cell line mass spectrometry (MS/MS) dataset, the tUTR
DB-based proteogenomic approach enabled the detection of 52 5′-UTR
and 9 3′-UTR peptides from 45 and 9 genes, respectively. The
identified UTR peptides were validated via high spectral similarity
with their synthetic peptides. The 5′-UTR peptides pointed
out alternative initiation sites with non-AUG start codons, which
exactly conformed to Kozak contexts of annotated initiation sites.
It is also worth noting that our approach can detect translated amino
acid sequences as well as provide evidence for UTR translation, while
ribosome profiling provides only the translation evidence. For previously
reported stop codon readthrough in MDH1 gene, we could confirm the
amino acid inserted during the readthrough. Data are available via
ProteomeXchange with identifier PXD016207.