High-throughput proteomics detection of novel splice isoforms in human platelets

Area of Science:

• Molecular Biology
• Genomics
• Proteomics

Background:

• Alternative splicing (AS) is a key regulatory mechanism in metazoans, producing diverse splice isoforms from most human genes.
• AS variants are linked to tissue specificity, development, and diseases, including cancer, highlighting their importance.
• Detecting splice variants offers insights into genomic complexity and potential biomarkers for disease diagnosis and prognosis.

Purpose of the Study:

• To develop and apply a proteomics-based strategy for identifying exon skip events, a common form of AS.
• To directly detect splice variants at the protein level using mass spectrometry data.
• To validate novel splice isoforms in human platelets and demonstrate the method's broad applicability.

Main Methods:

• A proteomics approach was employed to identify exon skipping events.
• A specialized database was created containing peptide sequences from all hypothetical human exon skip junctions.
• Tandem mass spectrometry (MS/MS) data was searched against this database for direct detection of exon skipping events.

Main Results:

• The study successfully applied the proteomics approach to human platelets.
• Novel splice isoforms of ITGA2, NPEPPS, and FH were identified and verified using mRNA-based methods.
• The methodology enables direct protein-level detection of alternative splicing events.

Conclusions:

• The developed proteomics approach offers a powerful tool for identifying alternative splicing events, particularly exon skipping.
• This method facilitates the discovery of novel splice variants with potential as disease-specific and prognostic biomarkers.
• The technique is versatile and can be applied to existing and new MS/MS datasets for comprehensive analysis of alternative splicing.