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

      Power, Karen A; McRedmond, James P; de Stefani, Andreas; Gallagher, William M; Gaora, Peadar O; UCD Conway Institute and UCD School of Biomolecular & Biomedical Sciences, UCD Conway Institute, University College Dublin, Belfield, Dublin, Ireland. (2009)
      Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS/MS datasets.