Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome

Area of Science:

• Proteomics
• Molecular Biology
• Genomics

Background:

• The functional consequences of alternative splicing events at the protein level are not well understood.
• Identifying protein isoforms arising from alternative splicing is challenging.

Purpose of the Study:

• To develop and apply an RNA sequencing-guided proteomics method to identify novel protein alternative splicing isoforms in the human proteome.
• To investigate the regulation and potential functions of these newly identified isoforms.

Main Methods:

• Constructed tissue-specific protein databases prioritizing splice junction pairs with high translational potential.
• Reanalyzed public mass spectrometry proteomics datasets using these custom databases.
• Applied the method to quantitative mass spectrometry data from differentiating stem cells.

Main Results:

• Identified over 1,500 noncanonical protein isoforms across 12 human tissues.
• Discovered approximately 400 novel protein sequences not present in existing databases (TrEMBL, RefSeq).
• Observed widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation.

Conclusions:

• Alternative splicing significantly expands proteome diversity, generating numerous undocumented protein isoforms.
• Alternative isoform regions frequently overlap with intrinsically disordered regions and post-translational modification sites, suggesting a role in protein function regulation.
• The developed RNA-seq guided proteomics approach is effective for discovering and studying alternative splicing isoforms and can be applied to various biological systems.