Identifying protein-coding genes in eukaryotic genomes remains a challenge in post-genome era due to the complex gene models. We applied a proteogenomics strategy to detect un-annotated protein-coding regions in mouse genome. High-accuracy tandem mass spectrometry (MS/MS) data from diverse mouse samples were generated by LTQ-Orbitrap mass spectrometer in house. Two searchable diagnostic proteomic datasets were constructed, one with all possible encoding exon junctions, and the other with all putative encoding exons, for the discovery of novel exon splicing events and novel uninterrupted protein-coding regions. Altogether 29,586 unique peptides were identified. Aligning backwards to the mouse genome, the translation of 4,471 annotated genes were validated by the known peptides; and 172 genic events were defined in mouse genome by the novel peptides. The approach in the current work can provide substantial evidences for eukaryote genome annotation in encoding genes.