Detection of Known and Novel Small Proteins in Pseudomonas stutzeri Using a Combination of Bottom-Up and Digest-Free Proteomics and Proteogenomics

Small proteins of
around 50 aa in length have been largely
overlooked
in genetic and biochemical assays due to the inherent challenges with
detecting and characterizing them. Recent discoveries of their critical
roles in many biological processes have led to an increased recognition
of the importance of small proteins for basic research and as potential
new drug targets. One example is CcoM, a 36 aa subunit of the cbb3-type oxidase that plays an essential role in adaptation
to oxygen-limited conditions in Pseudomonas stutzeri
(P. stutzeri), a model for the clinically relevant,
opportunistic pathogen Pseudomonas aeruginosa. However, as no comprehensive data were available in P. stutzeri, we devised an integrated, generic approach
to study small proteins more systematically. Using the first complete
genome as basis, we conducted bottom-up proteomics analyses and established
a digest-free, direct-sequencing proteomics approach to study cells
grown under aerobic and oxygen-limiting conditions. Finally, we also
applied a proteogenomics pipeline to identify missed protein-coding
genes. Overall, we identified 2921 known and 29 novel proteins, many
of which were differentially regulated. Among 176 small proteins 16
were novel. Direct sequencing, featuring a specialized precursor acquisition
scheme, exhibited advantages in the detection of small proteins with
higher (up to 100%) sequence coverage and more spectral counts, including
sequences with high proline content. Three novel small proteins, uniquely
identified by direct sequencing and not conserved beyond P. stutzeri, were predicted to form an operon with
a conserved protein and may represent de novo genes.
These data demonstrate the power of this combined approach to study
small proteins in P. stutzeri and show
its potential for other prokaryotes.