Human proteome analysis now requires an understanding of protein isoforms. Researchers from the University of New South Wales 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 analysed with the 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 non-isoform-specific proteotypic peptides could specifically identify many isoforms. In both the above cases, isoforms will be detectable with targeted MS/MS assays. Unfortunately, the analysis also revealed that some isoforms will be difficult to identify unambiguously as they do not have peptides that are sufficiently distinguishing. The researchers co-visualise mRNA isoforms and peptides in a genome browser to illustrate the above situations.

Overview of TranscriptCoder algorithm