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

Power KA, McRedmond JP, de Stefani A, Gallagher WM, Gaora PO - PLoS ONE (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: UCD Conway Institute and UCD School of Biomolecular & Biomedical Sciences, UCD Conway Institute, University College Dublin, Belfield, Dublin, Ireland.

ABSTRACT
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.

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Related in: MedlinePlus

Agarose gel electrophoresis of PCR products.Lanes 1–5 show products amplified from megakaryocyte cDNA; samples in lanes 6–10 contained template from platelets. Molecular size markers are in lane 11.
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pone-0005001-g006: Agarose gel electrophoresis of PCR products.Lanes 1–5 show products amplified from megakaryocyte cDNA; samples in lanes 6–10 contained template from platelets. Molecular size markers are in lane 11.

Mentions: PCR products of the expected sizes were observed in each case with cDNA derived both from platelets and from their precursors, megakaryocytes. The bands derived from platelet cDNA were excised and the sequence verified that the predicted products were obtained. It can be seen from Figure 6 that the megakaryocyte template produced a greater quantity of the amplicon in each case, reflective of the availability of template rather than an increased proportion of AS message in these cells.


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

Power KA, McRedmond JP, de Stefani A, Gallagher WM, Gaora PO - PLoS ONE (2009)

Agarose gel electrophoresis of PCR products.Lanes 1–5 show products amplified from megakaryocyte cDNA; samples in lanes 6–10 contained template from platelets. Molecular size markers are in lane 11.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654914&req=5

pone-0005001-g006: Agarose gel electrophoresis of PCR products.Lanes 1–5 show products amplified from megakaryocyte cDNA; samples in lanes 6–10 contained template from platelets. Molecular size markers are in lane 11.
Mentions: PCR products of the expected sizes were observed in each case with cDNA derived both from platelets and from their precursors, megakaryocytes. The bands derived from platelet cDNA were excised and the sequence verified that the predicted products were obtained. It can be seen from Figure 6 that the megakaryocyte template produced a greater quantity of the amplicon in each case, reflective of the availability of template rather than an increased proportion of AS message in these cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: UCD Conway Institute and UCD School of Biomolecular & Biomedical Sciences, UCD Conway Institute, University College Dublin, Belfield, Dublin, Ireland.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus