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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

Characteristics of the exon skip events detected in human platelets.(A) and (B) describe the distribution of the SkipE and IPI peptides respectively, across the different subcellular compartments for both resting and activated platelet samples.
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pone-0005001-g003: Characteristics of the exon skip events detected in human platelets.(A) and (B) describe the distribution of the SkipE and IPI peptides respectively, across the different subcellular compartments for both resting and activated platelet samples.

Mentions: The spatial distribution of AS identifications closely mirrors that of the IPI data with the exception of the releasate (Fig. 3a, b). In this case, more skips were found in the activated than in the resting samples for the AS data. Although the activation step was very brief, this may indicate a tendency towards diversification of the exported proteome in response to platelet activation. Functionally, this would be advantageous since these cells must interact with the milieu and other cell types but cannot mount a transcriptomic response to stimuli. All identified proteins in both SkipE and IPI data were mapped to KEGG pathways using Pathway-Express [27] (Table S3 and S4). In a typical MS/MS data analysis, protein identifications rely on multiple peptide identifications for any given protein. Since SkipE harbors isolated peptide sequences, we decided to focus further experiments on those AS events for which evidence of cognate gene expression was also obtained in the IPI analysis. Therefore, we constructed a list of 89 genes which represents the intersection of the AS and IPI datasets (Table 1).


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)

Characteristics of the exon skip events detected in human platelets.(A) and (B) describe the distribution of the SkipE and IPI peptides respectively, across the different subcellular compartments for both resting and activated platelet samples.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005001-g003: Characteristics of the exon skip events detected in human platelets.(A) and (B) describe the distribution of the SkipE and IPI peptides respectively, across the different subcellular compartments for both resting and activated platelet samples.
Mentions: The spatial distribution of AS identifications closely mirrors that of the IPI data with the exception of the releasate (Fig. 3a, b). In this case, more skips were found in the activated than in the resting samples for the AS data. Although the activation step was very brief, this may indicate a tendency towards diversification of the exported proteome in response to platelet activation. Functionally, this would be advantageous since these cells must interact with the milieu and other cell types but cannot mount a transcriptomic response to stimuli. All identified proteins in both SkipE and IPI data were mapped to KEGG pathways using Pathway-Express [27] (Table S3 and S4). In a typical MS/MS data analysis, protein identifications rely on multiple peptide identifications for any given protein. Since SkipE harbors isolated peptide sequences, we decided to focus further experiments on those AS events for which evidence of cognate gene expression was also obtained in the IPI analysis. Therefore, we constructed a list of 89 genes which represents the intersection of the AS and IPI datasets (Table 1).

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