Validation of mesenchymal stem cell transcripts assembled from RNA-Seq using proteomics data — ASN Events
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Validation of mesenchymal stem cell transcripts assembled from RNA-Seq using proteomics data (#205)

Aidan Tay 1 2 , Chi Pang 1 2 , Natalie Twine 1 2 , Gene Hart-Smith 1 2 , Linda Harkness 3 , Moustapha Kassem 3 , Marc Wilkins 1 2
  1. School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, Australia
  2. Systems Biology Initiative, The University of New South Wales, Sydney, New South Wales, Australia
  3. Department of Endocrinology and Metabolism, Odense University Hospital & University of Southern Denmark, Odense, Denmark
Alternative splicing of mRNA diversifies the function of human proteins, with tissue- and cell-specific protein isoforms being the most difficult to validate. While transcriptomic experiments enable the detection of many alternatively spliced transcripts, it is not known if these transcripts have protein-coding potential. We recently published the PG Nexus pipeline1, which facilitates high confidence validation of exons and exon-exon junctions of spliced transcripts by integrating transcriptomics and proteomics data. In this study, we applied PG Nexus towards the analysis of an undifferentiated human mesenchymal stem cell line and compared the number of protein isoforms validated using different protein sequence database, including public online databases and RNA-seq derived databases. With significant overlaps with other databases, we identified 8,011 exons and 3,824 splice junctions from 2,379 genes with the Ensembl database. The Ensembl database consistently outperformed the other data sources, but predicted open reading frames from RNA-seq derived transcripts were comparable, with only 6 less splice junctions validated. Using proteotypic and isoform-specific peptides, we validated 462 protein isoforms and a higher number is possible if we included multiple proteotypic peptides. Multiplexing proteotypic peptides in SRM assays or similar experiments will increase the confidence and coverage of protein isoform validation experiments.
  1. Pang, C. N.; Tay, A. P.; Aya, C.; Twine, N. A.; Harkness, L.; Hart-Smith, G.; Chia, S. Z.; Chen, Z.; Deshpande, N. P.; Kaakoush, N. O.; Mitchell, H. M.; Kassem, M.; Wilkins, M. R., Tools to covisualize and coanalyze proteomic data with genomes and transcriptomes: validation of genes and alternative mRNA splicing. J Proteome Res 2014, 13, (1), 84-98.