Revealing the small proteome of<i>Haloferax volcanii</i>by combining ribosome profiling and small-protein optimized mass spectrometry

Abstract

In contrast to extensively studied prokaryotic 'small' transcriptomes (encompassing all small noncoding RNAs), small proteomes (here defined as including proteins ≤70 aa) are only now entering the limelight. The absence of a complete small protein catalogue in most prokaryotes precludes our understanding of how these molecules affect physiology. So far, archaeal genomes have not yet been analyzed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental data from small protein-optimized mass spectrometry (MS) and ribosome profiling (Ribo-seq), to generate a high confidence inventory of small proteins in the model archaeon <i>Haloferax volcanii</i>. We demonstrate by MS and Ribo-seq that 67% of the 317 annotated small open reading frames (sORFs) are translated under standard growth conditions. Furthermore, annotation-independent analysis of Ribo-seq data showed ribosomal engagement for 47 novel sORFs in intergenic regions. A total of seven of these were also detected by proteomics, in addition to an eighth novel small protein solely identified by MS. We also provide independent experimental evidence <i>in vivo</i> for the translation of 12 sORFs (annotated and novel) using epitope tagging and western blotting, underlining the validity of our identification scheme. Several novel sORFs are conserved in <i>Haloferax</i> species and might have important functions. Based on our findings, we conclude that the small proteome of <i>H. volcanii</i> is larger than previously appreciated, and that combining MS with Ribo-seq is a powerful approach for the discovery of novel small protein coding genes in archaea.