Optimized Proteomics Workflow for the Detection of Small Proteins.

Area of Science:

• Proteomics
• Molecular Biology
• Bacterial Physiology

Background:

• Small open reading frame encoded proteins (SEPs) play vital roles in prokaryotes and eukaryotes, including bacterial signaling and virulence.
• Traditional proteomics methods often underrepresent SEPs due to biases favoring larger proteins, limiting our understanding of their functions.

Purpose of the Study:

• To develop and validate an optimized proteomics workflow for the enhanced identification and characterization of small proteins (SEPs).
• To improve the detection rate of SEPs in bacterial proteome studies compared to conventional protocols.

Main Methods:

• Optimization of small protein enrichment using small-pore-sized solid-phase material.
• Adjustment of proteolytic digestion protocols, including the use of alternative proteases to trypsin.
• Application of an integrated proteogenomics search database and rigorous validation strategies.

Main Results:

• The optimized workflow increased SEP identification by a factor of two.
• 210 annotated proteins up to 100 amino acids (aa) were detected in *Bacillus subtilis*, with 16 proteins shorter than 51 aa.
• Three novel, unannotated SEPs (21, 26, and 42 aa) were confidently identified.

Conclusions:

• The developed proteomics workflow significantly enhances the discovery of small proteins, including previously undiscovered SEPs.
• This advancement provides a more comprehensive view of the proteome, crucial for understanding bacterial biology and function.