Discovery of Novel Genes and Gene Isoforms by Integrating Transcriptomic and Proteomic Profiling from Mouse Liver

Comprehensively
identifying gene expression in both transcriptomic
and proteomic levels of one tissue is a prerequisite for a deeper
understanding of its biological functions. Alternative splicing and
RNA editing, two main forms of transcriptional processing, play important
roles in transcriptome and proteome diversity and result in multiple
isoforms for one gene, which are hard to identify by mass spectrometry
(MS)-based proteomics approach due to the relative lack of isoform
information in standard protein databases. In our study, we employed
MS and RNA-Seq in parallel into mouse liver tissue and captured a
considerable catalogue of both transcripts and proteins that, respectively,
covered 60 and 34% of protein-coding genes in Ensembl. We then developed
a bioinformatics workflow for building a customized protein database
that for the first time included new splicing-derived peptides and
RNA-editing-caused peptide variants, allowing us to more completely
identify protein isoforms. Using this experimentally determined database,
we totally identified 150 peptides not present in standard biological
databases at false discovery rate of <1%, corresponding to 72 novel
splicing isoforms, 43 new genetic regions, and 15 RNA-editing sites.
Of these, 11 randomly selected novel events passed experimental verification
by PCR and Sanger sequencing. New discoveries of gene products with
high confidence in two omics levels demonstrated the robustness and
effectiveness of our approach and its potential application into improve
genome annotation. All the MS data have been deposited to the iProx
(http://ww.iprox.org) with the identifier IPX00003601.