Abstract
Ribosome profiling has revealed translation outside canonical coding sequences, including translation of short upstream ORFs, long noncoding RNAs, overlapping ORFs, ORFs in UTRs, or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling, and CRISPR-based screens showed that hundreds of ORFs derived from noncoding transcripts produce (micro)proteins, whereas other studies failed to find evidence for such types of noncanonical translation products. Here, we attempted to discover translation products from noncoding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. We show that, theoretically our strategy facilitates the detection of translation events of transcripts from noncoding regions but experimentally only find 19 peptides that might originate from such translation events. Finally, Virotrap-based interactome analysis of two N-terminal proteoforms originating from noncoding regions showed the functional potential of these novel proteins.