Human proteomic databases required for MS peptide identification are frequently updated and carefully curated, yet are still incomplete because it has been challenging to acquire every protein sequence from the diverse assemblage of proteoforms expressed in every tissue and cell type. In particular, alternative splicing has been shown to be a major source of this cell-specific proteomic variation. Many new alternative splice forms have been detected at the transcript level using next generation sequencing methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of next generation sequencing methods, 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. Eighty million paired-end Illumina reads and ∼500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6810 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. Based on the RefSeq gene models, we detected 136,123 annotated and 144,818 unannotated transcript junctions. Of those, 24,834 unannotated junctions passed various quality filters (e.g. minimum read depth) and these entries were translated into 33,589 polypeptide sequences and used for database searching. We discovered 57 splice junction peptides not present in the Uniprot-Trembl proteomic database comprising an array of different splicing events, including skipped exons, alternative donors and acceptors, and noncanonical transcriptional start sites. To our knowledge this is the first example of using sample-specific RNA-Seq data to create a splice-junction database and discover new peptides resulting from alternative splicing.