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, 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 are investigated. In total, 45 SEP with at least one high confidence (FDR <1%) unique peptide and five consecutive b‐ or y‐ions in the MS2 spectrum are identified. The staining methods provide complementary data. The highest number of SEP are identified in the samples stained with Coomassie brilliant blue. However, the highest quality of the identified SEP is achieved in the samples without staining. These comprehensive data sets demonstrate that in‐gel digestion is well suited for the identification of SEP.

中文翻译：

---

不同 SDS-PAGE 凝胶染色方法的互补性，用于识别短开放阅读框编码的肽。

短开放阅读框编码肽 (SEP) 已在生命的所有领域中得到鉴定，并预计参与许多生化过程，然而，对于绝大多数 SEP，它们的生物学功能仍然未知。优化的方法必须用于 SEP 的质谱分析，因为自下而上的蛋白质组学的传统方法显示出对小蛋白质的偏见。在这里，在进行 LC-MS/MS 分析后进行凝胶内消化之前，SDS-PAGE 凝胶的不同染色方法用于鉴定古细菌Methanosarcina mazei 中的 SEP被调查。总共鉴定了 45 个具有至少一个高置信度 (FDR <1%) 独特肽段和 MS2 质谱图中连续 5 个 b 或 y 离子的 SEP。染色方法提供了补充数据。在考马斯亮蓝染色的样品中鉴定出最高数量的 SEP。然而，在未染色的样品中实现了已识别 SEP 的最高质量。这些综合数据集表明凝胶内消化非常适合鉴定 SEP。