Optimized Proteomics Workflow for the Detection of Small Proteins

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 enhanced identification 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 number
of identified SEPs by a factor of 2, and utilization of alternative
proteases to trypsin reduced the spectral counts for larger proteins.
The application of the optimized protocol allowed the detection of
210 already annotated proteins up to 100 amino acids (aa) length,
including 16 proteins below 51 aa in the Gram-positive model organism Bacillus subtilis. Moreover, 12% of all identified
proteins were up to 100 aa, which is a significantly larger fraction
than that reported in studies involving traditional proteomics workflows.
Finally, the application of an integrated proteogenomics search database
and extensive subsequent validation resulted in the confident identification
of three novel, not yet annotated, SEPs, which are 21, 26, and 42
aa long.