@Article{Hadjeras_Bartel_Maier-Revea_the_small-2023,
  author =	 {Hadjeras, Lydia and Bartel, Jurgen and Maier, 
                  Lisa-Katharina and Maass, Sandra and Vogel, Verena and 
                  Svensson, Sarah L. and Eggenhofer, Florian and Gelhausen, 
                  Rick and Muller, Teresa and Alkhnbashi, Omer S. and 
                  Backofen, Rolf and Becher, Dorte and Sharma, Cynthia M. and 
                  Marchfelder, Anita},
  title =	 {Revealing the small proteome of {Haloferax} volcanii by 
                  combining ribosome profiling and small-protein optimized 
                  mass spectrometry},
  journal =	 {Microlife},
  year =	 {2023},
  volume =	 {4},
  number =	 {},
  pages =	 {uqad001},
  user =	 {gelhausr},
  pmid =	 {37223747},
  doi = 	 {10.1093/femsml/uqad001},
  issn = 	 {2633-6693},
  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 Haloferax volcanii. 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 in vivo 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 
                  Haloferax species and might have important functions. Based 
                  on our findings, we conclude that the small proteome of H. 
                  volcanii 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.}
}