title: Proteomic Validation of Transcript Isoforms, Including Those Assembled from RNA-Seq Data.
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).
authors.affiliation: Systems Biology Initiative, The University of New South Wales , Sydney, New South Wales 2052, Australia.|School of Biotechnology and Biomolecular Sciences, The University of New South Wales , Sydney, New South Wales 2052, Australia.
authors.forename: Aidan P
authors.initials: AP
authors.lastname: Tay
authors.affiliation: Systems Biology Initiative, The University of New South Wales , Sydney, New South Wales 2052, Australia.|School of Biotechnology and Biomolecular Sciences, The University of New South Wales , Sydney, New South Wales 2052, Australia.
authors.forename: Chi Nam Ignatius
authors.initials: CN
authors.lastname: Pang
authors.affiliation: Systems Biology Initiative, The University of New South Wales , Sydney, New South Wales 2052, Australia.|School of Biotechnology and Biomolecular Sciences, The University of New South Wales , Sydney, New South Wales 2052, Australia.
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Twine
authors.affiliation: Systems Biology Initiative, The University of New South Wales , Sydney, New South Wales 2052, Australia.|School of Biotechnology and Biomolecular Sciences, The University of New South Wales , Sydney, New South Wales 2052, Australia.
authors.forename: Gene
authors.initials: G
authors.lastname: Hart-Smith
authors.affiliation: Endocrine Research Laboratory (KMEB), Department of Endocrinology and Metabolism, Odense University Hospital & University of Southern Denmark , Odense 5230, Denmark.
authors.forename: Linda
authors.initials: L
authors.lastname: Harkness
authors.affiliation: Endocrine Research Laboratory (KMEB), Department of Endocrinology and Metabolism, Odense University Hospital & University of Southern Denmark , Odense 5230, Denmark.
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
authors.affiliation: Systems Biology Initiative, The University of New South Wales , Sydney, New South Wales 2052, Australia.|School of Biotechnology and Biomolecular Sciences, The University of New South Wales , Sydney, New South Wales 2052, Australia.
authors.forename: Marc R
authors.initials: MR
authors.lastname: Wilkins
mesh_terms: D002478:Cells, Cultured; D003062:Codon; D005091:Exons; D006801:Humans; D016366:Open Reading Frames; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D040901:Proteomics; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D017423:Sequence Analysis, RNA; D053719:Tandem Mass Spectrometry
chemical_list: D003062:Codon; D020033:Protein Isoforms; D012333:RNA, Messenger
keywords: RNA-seq; alternative splicing; isoform-specific peptides; mesenchymal stem cell; proteotypic peptides; splice-junction peptides