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J Proteome Res. 2015 Sep 4;14(9):3541-54. doi: 10.1021/pr5011394. Epub 2015 May 20.

Proteomic Validation of Transcript Isoforms, Including Those Assembled from RNA-Seq Data.

Author information

1
Systems Biology Initiative, The University of New South Wales , Sydney, New South Wales 2052, Australia.
2
School of Biotechnology and Biomolecular Sciences, The University of New South Wales , Sydney, New South Wales 2052, Australia.
3
Endocrine Research Laboratory (KMEB), Department of Endocrinology and Metabolism, Odense University Hospital & University of Southern Denmark , Odense 5230, Denmark.

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).

KEYWORDS:

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

PMID:
25961807
DOI:
10.1021/pr5011394
[Indexed for MEDLINE]

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