Dataset Information

Optimised Workflow for the Proteomic Detection of Small Proteins

ABSTRACT: Small open reading frame encoded proteins (SEPs) gained increasing interest during the last years due to their broad range of important functions in both, prokaryotes and in eukaryotes. In bacteria, signalling, virulence or regulation of enzyme activities have been associated with this protein class. Nonetheless, the number of SEPs detected in large scale proteome studies is often low as classical methods are biased towards the identification of larger proteins. In the accompanying manuscript, we present a workflow that allows enhanced identification rates of small proteins compared to traditional protocols. For this aim, the steps of small protein enrichment, proteolytic digest and database search were reviewed and adjusted to the special requirement of SEPs. Enrichment by the use of small pore sized solid phase material increased the absolute numbers of identified SEPs by a factor of two and the application of alternative proteases to trypsin reduced spectral counts for larger proteins. The application of an integrated proteogenomics search database (iPtgxDB) in combination with the optimized protocol allowed the detection of several proteins not yet annotated in the reference genome of Bacillus subtilis. Finally, the identification of some of these missed proteins was validated by comparing their identifying spectra to those of synthetic peptides.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Bacillus Subtilis Subsp. Subtilis Str. 168

SUBMITTER: Juergen Bartel

LAB HEAD: Dörte Becher

PROVIDER: PXD017416 | Pride | 2020-10-12

REPOSITORIES: Pride

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Optimized Proteomics Workflow for the Detection of Small Proteins.

Bartel Jürgen J Varadarajan Adithi R AR Varadarajan Adithi R AR Sura Thomas T Ahrens Christian H CH Maaß Sandra S Becher Dörte D

Journal of proteome research 20200902 10

Small open reading frame encoded proteins (SEPs) gained increasing interest during the last few years because of their broad range of important functions in both prokaryotes and eukaryotes. In bacteria, signaling, virulence, and regulation of enzyme activities have been associated with SEPs. Nonetheless, the number of SEPs detected in large-scale proteome studies is often low as classical methods are biased toward the identification of larger proteins. Here, we present a workflow that allows enh ...[more]

PMID: 32812434

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Project description:Many small open reading frames (smORFs) embedded in lncRNA transcripts have been shown to encode biologically functional polypeptides (smORFs-encoded polypeptides, SEPs) in different organisms. Despite significant advances in genomics, bioinformatics and proteomics that largely enabled the discovery of novel SEPs, their identification across different biological samples is still hampered by their poor predictability, diminutive size and low relative abundance. Here, we take advantage of NONCODE, a repository containing the most complete collection and annotation of lncRNA transcripts from different species, to build a novel database that attempts to maximize a collection of SEPs from human and mouse lncRNA transcripts. In order to further improve SEP discovery, we implemented two effective and complementary polypeptide enrichment strategies, 30 kDa MWCO filter and C8 SPE column. These combined strategies enabled us to discover 357 and 409 SEPs from, respectively, 8 human cell lines, and 3 mouse cell lines and 8 mouse tissues. Importantly, nineteen of the identified SEPs were then verified through in-vitro expression, immunoblotting, parallel reaction monitoring (PRM) and synthetic peptides. Subsequent bioinformatic analysis revealed that some of the physical and chemical properties of these novel SEPs, including amino acid composition and codon usage, are different from those commonly found in canonical proteins. Intriguingly, nearly 65% of the identified SEPs were found to be initiated with non-AUG start codons. Overall, the strategy presented in this study encompasses an efficient workflow that enabled us to identify 766 novel SEPs across multiple cell lines and tissues, which probably represents the largest number of SEPs detected by mass spectrometry reported to date. These novel SEPs might not only provide new clues for the annotation of noncoding elements in the genome but can also serve as a valuable resource for the functional characterization of individual SEPs.