Proteomics

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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]

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