@article{Hadjeras_Bartel_Maier-Revea_the_small-2023,
author = {Hadjeras, Lydia and Bartel, Jürgen and Maier, 
          Lisa-Katharina and Maaß, Sandra and Vogel, Verena and 
          Svensson, Sarah L. and Eggenhofer, Florian and Gelhausen, 
          Rick and Müller, Teresa and Alkhnbashi, Omer S. and 
          Backofen, Rolf and Becher, Dörte 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},
doi = {10.1093/femsml/uqad001},
volume = {4},
user = {gelhausr},
pmid = {37223747},
pages = {uqad001},
number = {},
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.}
}