IS-PRM-based peptide targeting informed by long-read sequencing for alternative proteome detection

Area of Science:

• Proteomics
• Transcriptomics
• Molecular Biology

Background:

• Alternative splicing generates diverse protein isoforms, but their functional relevance and detection remain challenging.
• Existing methods struggle to identify scarce, isoform-specific peptides due to technical limitations.
• Targeted mass spectrometry (MS) strategies like internal standard parallel reaction monitoring (IS-PRM) offer sensitive detection but haven't been applied to novel peptide discovery.

Approach:

• Developed a targeted proteogenomic approach (LRP-IS-PRM) integrating long-read RNA sequencing (LR RNAseq) with IS-PRM (Tomahto).
• Used LR RNAseq to predict potential protein isoforms and generate synthetic 'trigger' peptides.
• Spiked labeled synthetic peptides into cell digests to detect corresponding endogenous 'target' peptides via IS-PRM.

Key Points:

• Identified 54 resolved protein isoforms from 43 genes, including five previously unannotated isoforms.
• The LRP-IS-PRM method increased isoform detectability by 3.6-fold compared to data-dependent acquisition (DDA) mode.
• Successfully generated protein-level evidence for alternative isoforms, aiding future functional studies.

Conclusions:

• LRP-IS-PRM provides a novel, sensitive modality for confirming protein isoforms predicted by transcriptomic data.
• This approach overcomes limitations in detecting isoform-specific peptides, advancing the study of proteomic diversity.
• Enables the critical first step in characterizing novel protein isoforms for functional and clinical applications.