Proteomic validation of transcript isoforms, including those assembled from RNA-Seq data

Aidan P. Tay, Chi Nam Ignatius Pang, Natalie A. Twine, Gene Hart-Smith, Linda Harkness, Moustapha Kassem, Marc R. Wilkins*

*Corresponding author for this work

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Human proteome analysis now requires an understanding of protein isoforms. We recently published the PG Nexus pipeline, which facilitates high confidence validation of exons and splice junctions by integrating genomics and proteomics data. Here we comprehensively explore how RNA-seq transcriptomics data, and proteomic analysis of the same sample, can identify protein isoforms. RNA-seq data from human mesenchymal (hMSC) stem cells were analyzed with our new TranscriptCoder tool to generate a database of protein isoform sequences. MS/MS data from matching hMSC samples were then matched against the TranscriptCoder-derived database, along with Ensembl and the neXtProt database. Querying the TranscriptCoder-derived or Ensembl database could unambiguously identify ∼450 protein isoforms, with isoform-specific proteotypic peptides, including candidate hMSC-specific isoforms for the genes DPYSL2 and FXR1. Where isoform-specific peptides did not exist, groups of nonisoform-specific proteotypic peptides could specifically identify many isoforms. In both the above cases, isoforms will be detectable with targeted MS/MS assays. Unfortunately, our analysis also revealed that some isoforms will be difficult to identify unambiguously as they do not have peptides that are sufficiently distinguishing. We covisualize mRNA isoforms and peptides in a genome browser to illustrate the above situations. Mass spectrometry data is available via ProteomeXchange (PXD001449).

Original languageEnglish
Pages (from-to)3541-3554
Number of pages14
JournalJournal of Proteome Research
Volume14
Issue number9
DOIs
Publication statusPublished - 4 Sep 2015
Externally publishedYes

Keywords

  • alternative splicing
  • isoform-specific peptides
  • mesenchymal stem cell
  • proteotypic peptides
  • RNA-seq
  • splice-junction peptides

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  • Cite this

    Tay, A. P., Pang, C. N. I., Twine, N. A., Hart-Smith, G., Harkness, L., Kassem, M., & Wilkins, M. R. (2015). Proteomic validation of transcript isoforms, including those assembled from RNA-Seq data. Journal of Proteome Research, 14(9), 3541-3554. https://doi.org/10.1021/pr5011394