Mapping Start Codons of Small Open Reading Frames by N-Terminomics Approach

Area of Science:

• Molecular Biology
• Proteomics
• Genomics

Background:

• Small open reading frame-encoded peptides (SEPs) are crucial proteins, but their characterization is limited by reliance on bioinformatics predictions.
• Current methods often incorrectly assume AUG as the start codon for SEPs, overlooking non-canonical initiation sites.

Purpose of the Study:

• To systematically analyze and accurately define the N-terminal sequences and start sites of SEPs using chemical labeling.
• To improve the identification and characterization of SEPs, including those initiated by non-canonical codons.

Main Methods:

• Chemical labeling techniques were employed to analyze SEP N-terminal sequences.
• Optimized enrichment strategies using ACN precipitation, heating precipitation, and Hexadhexaldehyde were developed.
• Mass spectrometry was used to identify SEPs and their N-terminal sequences.

Main Results:

• 128 SEPs with 131 N-terminal sequences were identified, with two-thirds being novel.
• Most novel N-termini started within amino acids 11-31 of the original sequence, with some resulting from proteolysis or signal peptide removal.
• Chemical labeling corrected several SEPs' start codons to non-AUG sites, with one novel start codon validated experimentally.

Conclusions:

• Chemical labeling provides a robust approach for identifying accurate start codons of sORFs and the true N-termini of SEPs.
• This methodology enhances the understanding of SEP diversity and function, particularly for those with non-canonical initiation.
• The findings facilitate more precise characterization of SEPs and their biological roles.