Updated project metadata. Short open reading frame-encoded peptides (SEP) have been identified across all domains of life and are predicted to be involved in many biochemical processes, however, for the vast majority of SEP their biological function is still unknown. Special methodologies have to be used for the mass spectrometric analysis of SEP, because traditional methods of bottom-up proteomics show a bias against small proteins. Here, we investigated the influence of different staining methods for SDS-PAGE gels prior in-gel digestion following LC-MS/MS analysis for the identification of SEP in the archaeon Methanosarcina mazei. In total, we identified 82 SEP with at least one high confidence (FDR<1 %) unique peptide, of which 45 also met a stricter ion series-based quality criteria. The staining methods provided complementary data that allowed for a range of different SEP to be identified. The highest number of SEP were identified in the samples stained with Coomassie brilliant blue. However, the highest quality of the identified SEP was achieved in the samples without staining. These data demonstrate that in-gel digestion is amenable and well suited for the identification of SEP, and that this classical technique may provide further insights into the world of SEP.