Using RNA-Seq to create sample-specific proteomic databases that enable mass spectrometric discovery of splice junction peptides
Many new alternative splice forms have been detected at the transcript level using next generation sequencing (NGS) methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated.  Leveraging the unprecedented capabilities of NGS, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splice-junction peptides. Results: Eighty million paired-end Illumina reads and ~500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6,810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching. Overall design: Jurkat T-cell mRNA was analyzed on an Illumina HiSeq2000.  ~80 million paired end reads (2x200bp, ~350bp lengths) were collected.