Determining Alternative Protein Isoform Expression Using RNA Sequencing and Mass Spectrometry

Area of Science:

• Genomics and Proteomics
• Molecular Biology

Background:

• Alternative splicing significantly increases the coding potential of the human genome.
• The number of translated alternative transcripts and their functional significance remain largely unverified experimentally.

Purpose of the Study:

• To describe a protocol for identifying alternative protein isoforms through integrated transcriptomics and proteomics.
• To provide a method applicable to both new and existing biological datasets.

Main Methods:

• Combined analysis of RNA sequencing (RNA-seq) and mass spectrometry (MS) data.
• Workflow adaptable for custom-generated data from matching samples.
• Reanalysis of publicly available transcriptomics and proteomics datasets.

Main Results:

• The protocol enables the identification of alternative splicing isoforms present at the protein level.
• Demonstrates the feasibility of integrating transcriptomic and proteomic data for isoform discovery.
• Provides a framework for experimental verification of functional alternative transcripts.

Conclusions:

• The integrated RNA-seq and MS approach is a powerful tool for discovering translated alternative isoforms.
• This method advances our understanding of the functional proteome by characterizing previously unannotated protein variants.
• Experimental verification of alternative splicing's functional impact is crucial and facilitated by this protocol.