Exploring the Alternative Proteome with OpenProt and Mass Spectrometry.

Area of Science:

• Proteogenomics
• Transcriptomics
• Bioinformatics

Background:

• Proteogenomics reveals translation from unannotated open reading frames (ORFs) in messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs).
• Existing databases like UniProt, RefSeq, and Ensembl primarily annotate canonical proteins, leaving many translated ORFs undiscovered.
• The discovery of noncanonical proteins, including novel isoforms and alternative proteins, is crucial for a comprehensive understanding of the proteome.

Purpose of the Study:

• To introduce OpenProt, a comprehensive database for annotating all ORFs (≥30 codons) within a species' transcriptome.
• To describe methods for identifying multiple ORFs within genes and transcripts.
• To explain the utilization of OpenProt for detecting novel isoforms and alternative proteins via mass spectrometry-based proteomics.

Main Methods:

• Transcriptome-wide annotation of all ORFs with a minimum length of 30 codons.
• Classification of annotated proteins into reference (canonical) and noncanonical types (novel isoforms, alternative proteins).
• Utilizing OpenProt database queries and mass spectrometry data for protein identification and validation.

Main Results:

• OpenProt successfully annotates a vast number of ORFs, significantly expanding the known proteome.
• The database distinguishes between novel isoforms with homology to known proteins and novel alternative proteins lacking homology.
• The described methods enable the detection and characterization of these noncanonical proteins in proteomic studies.

Conclusions:

• OpenProt provides a valuable resource for exploring the unannotated proteome derived from ORFs.
• The platform facilitates the discovery of novel protein isoforms and entirely new proteins, advancing proteogenomic research.
• Integrating OpenProt with mass spectrometry-based proteomics enhances the identification of previously undiscovered protein-coding elements.