PROTEOFORMER

PROTEOFORMER automates the processing of ribosome profiling (RIBO-seq) data to generate genome-wide translation product candidates that enhance mass spectrometry-based proteoform identification.

Key Features:

• Ribosome Profiling Data Processing: Automated processing of RIBO-seq data involving sequencing of ribosome-protected mRNA fragments.
• P-site Offset Calculation: Calculation of P-site offsets during mapping to improve positional accuracy of ribosome occupancy.
• Translation Initiation Site Calling: Algorithm for delineating open reading frames (ORFs) of all translation products.
• Alternative Proteoform Calling Strategies: Two alternative strategies for calling proteoforms that can be used alongside one another and combined with reference sequences from UniProt.
• Comprehensive Data Pre-exploration: Extended pipeline output features for thorough data exploration prior to analysis.
• Prosit Integration: Optional inclusion of Prosit, a deep neural network strategy that adds extra fragmentation spectrum intensity features for improved spectrum matching validation.
• MaxQuant Compatibility: Generated search spaces can be searched with MaxQuant for mass spectrometry data analysis.

Scientific Applications:

• New Proteoform Discovery: Identification and validation of different categories of new proteoforms including translation products of up- and downstream open reading frames, 5' and 3' extended and truncated proteoforms, single amino acid variants, splice variants, and translation products of noncoding regions.
• Proteome Coverage Enhancement: Increased overall protein identification rates with 3% and 11% (improved and new identifications) for human and mouse proteomes.
• 5'-Extended Proteoform Detection: Proteome-wide detection of 5'-extended proteoforms, upstream ORF translation, and near-cognate translation start sites.
• Ribosome Profiling-Driven Proteogenomics: Validation of spectrum matches of newly identified proteoforms with elevated stringency through integrated ribosome occupancy and mass spectrometry analysis.

Methodology:

The pipeline performs genome-wide construction of candidate translation products based on ribosome occupancy, including P-site offset calculation during mapping, translation initiation site calling for ORF delineation, and two alternative proteoform calling strategies that are combined with UniProt reference sequences for mass spectrometry search space generation.