Revealing the small proteome of Haloferax volcanii by ribosome profiling and small-protein optimised mass spectrometry
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ABSTRACT:
ORGANISM(S): Haloferax volcanii
PROVIDER: GSE208086 | GEO | 2023/01/18
REPOSITORIES: GEO
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Project description:The small proteome has already been well explored in eukaryal and bacterial species, but so far, archaeal genomes have not yet been analysed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental information 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. Translation was demonstrated for 67% of the annotated small coding sequences by both methods. Annotation-independent data analysis allowed for the prediction of 47 sites of ribosomal engagement outside known coding regions by Ribo-seq, seven of whom correspond to the eight un-annotated small proteins identified by a similar independent analysis of proteomic data. We also present independent evidence in vivo for the translation of a subset of small proteins (comprising both previously annotated and newly identified), underlining the validity of our identification scheme. Moreover, several of these translated sORFs are conserved in Haloferax and might have important functions. Based on our findings, we conclude that the small proteome of H. volcanii is larger than previously expected and that the combined use of mass spectrometry to detect protein presence with Ribo-seq to inform on translation is a powerful tool for the discovery of new small protein-coding genes in diverse organisms. This data-set contains the search results obtained from an MS-Fragger search against a reference-genome derived database.
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Project description:The small proteome has already been well explored in eukaryal and bacterial species, but so far, archaeal genomes have not yet been analysed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental information 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. Translation was demonstrated for 67% of the annotated small coding sequences by both methods. Annotation-independent data analysis allowed for the prediction of 47 sites of ribosomal engagement outside known coding regions by Ribo-seq, seven of whom correspond to the eight un-annotated small proteins identified by a similar independent analysis of proteomic data. We also present independent evidence in vivo for the translation of a subset of small proteins (comprising both previously annotated and newly identified), underlining the validity of our identification scheme. Moreover, several of these translated sORFs are conserved in Haloferax and might have important functions. Based on our findings, we conclude that the small proteome of H. volcanii is larger than previously expected and that the combined use of mass spectrometry to detect protein presence with Ribo-seq to inform on translation is a powerful tool for the discovery of new small protein-coding genes in diverse organisms. This data-set contains the search results obtained from an MS-Fragger search against six-frame genome translation-derived database that were mapped to the genome by Stephan Fuchs “Salt & Pepper” software suite for bacterial proteogenomics.
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