Optimised Workflow for the Proteomic Detection of Small Proteins

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.