IS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection

Area of Science:

• Proteogenomics
• Mass Spectrometry
• Transcriptomics
• Alternative Splicing

Background:

• Alternative splicing generates diverse transcript isoforms, but their translation into functional proteins is often unconfirmed.
• Detecting isoform-specific peptides is challenging due to low abundance and technical limitations, leaving many protein isoforms uncharted.
• Targeted mass spectrometry (MS) strategies like internal standard parallel reaction monitoring (IS-PRM) offer sensitive detection but haven't been used for novel peptide confirmation.

Purpose of the Study:

• To develop and validate a targeted proteogenomic approach for confirming novel protein isoforms.
• To leverage long-read RNA sequencing (lrRNA-seq) data to predict and detect isoform-specific peptides.
• To assess the performance of the IS-PRM method, termed Tomahto, for identifying previously unannotated protein isoforms.

Main Methods:

• A proteogenomic strategy integrating sample-matched lrRNA-seq data with targeted mass spectrometry (Tomahto).
• lrRNA-seq data informed the design of synthetic 'trigger' peptides for 192 isoform-specific peptides across 55 genes.
• Super heavy tandem mass tags (TMT) labeled synthetic peptides were spiked into TMT-labeled cell digests for IS-PRM analysis.

Main Results:

• The Tomahto method increased isoform detectability by 3.6-fold compared to data-dependent acquisition (DDA) mode.
• Five previously unannotated protein isoforms were identified.
• Protein expression was confirmed for 54 resolved isoforms across 43 genes.

Conclusions:

• The lrRNA-seq-informed Tomahto approach provides a novel modality for generating protein-level evidence of alternative isoforms.
• This method is crucial for advancing functional studies and developing clinical assays for protein isoforms.
• The study demonstrates the power of integrating transcriptomic and proteomic data for comprehensive isoform discovery.