Complementarity of Different SDS-PAGE Gel Staining Methods for the Identification of Short Open Reading Frame-encoded Peptides.

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. Optimized 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 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 45 SEP with at least one high confidence (FDR<1 %) unique peptide and five consecutive b- or y-ions in the MS2 spectrum. The staining methods provided complementary data. 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 comprehensive data sets demonstrate that in-gel digestion is well suited for the identification of SEP. This article is protected by copyright. All rights reserved.