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

Small proteins ofaround 50 aa in length have been largelyoverlookedin genetic and biochemical assays due to the inherent challenges withdetecting and characterizing them. Recent discoveries of their criticalroles in many biological processes have led to an increased recognitionof the importance of small proteins for basic research and as potentialnew drug targets. One example is CcoM, a 36 aa subunit of the cbb3-type oxidase that plays an essential role in adaptationto 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 approachto study small proteins more systematically. Using the first completegenome as basis, we conducted bottom-up proteomics analyses and establisheda digest-free, direct-sequencing proteomics approach to study cellsgrown under aerobic and oxygen-limiting conditions. Finally, we alsoapplied a proteogenomics pipeline to identify missed protein-codinggenes. Overall, we identified 2921 known and 29 novel proteins, manyof which were differentially regulated. Among 176 small proteins 16were novel. Direct sequencing, featuring a specialized precursor acquisitionscheme, exhibited advantages in the detection of small proteins withhigher (up to 100%) sequence coverage and more spectral counts, includingsequences with high proline content. Three novel small proteins, uniquelyidentified by direct sequencing and not conserved beyond P. stutzeri, were predicted to form an operon witha conserved protein and may represent de novo genes.These data demonstrate the power of this combined approach to studysmall proteins in P. stutzeri and showits potential for other prokaryotes.

Automated summary

Small proteins have been overlooked in genetic and biochemical assays, but recent discoveries have highlighted their importance and potential as drug targets. The authors used an integrated approach to study small proteins in Pseudomonas stutzeri and identified known and novel proteins. This study demonstrates the power of the combined approach and its potential for studying small proteins in other prokaryotes.