Ortho-proteogenomics: multiple proteomes investigation through orthology and a new MS-based protocol

Area of Science:

• Genomics
• Proteomics
• Bioinformatics

Background:

• Advancements in sequencing generate vast numbers of genomes, often relying on in silico gene prediction.
• Predicted gene repertoires frequently contain errors, especially in start codons, impacting downstream biological research.
• Accurate genome annotation is crucial for understanding biological functions and pathways.

Purpose of the Study:

• To develop and present a novel "ortho-proteogenomic" approach for simultaneous annotation refinement of multiple genomes.
• To improve the accuracy of predicted protein sequences and identify previously missed genes.
• To validate the approach using the Mycobacterium genus.

Main Methods:

• The study combined comparative genomics with a novel proteomic protocol.
• The proteomic protocol enabled the simultaneous characterization of N-terminal and internal peptides.
• The strategy was applied to the Mycobacterium genus, using Mycobacterium smegmatis as a reference.

Main Results:

• The ortho-proteogenomic approach identified 946 distinct proteins, including 443 characterized N-termini.
• Experimental data led to the correction of 19% of predicted start codons.
• The study identified 29 previously unannotated proteins and curated 4328 sequences across 16 other Mycobacterium proteomes.

Conclusions:

• The ortho-proteogenomic approach significantly enhances the accuracy of genome annotation.
• This strategy effectively refines protein predictions, corrects start codon errors, and discovers novel proteins.
• The method provides a robust framework for improving proteome quality across multiple related genomes.