Zhang Tingting, Cheng Jian, Li Jiao, Ye Zixia, Li Na, Wang Jifeng, Yang Xiaojuan, Peng Yong
Center for Molecular Oncology, Frontiers Science Center for Disease-related Molecular Network, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China.
Department of Neurosurgery West China Hospital, Sichuan University, Chengdu, China.
Mol Cell Proteomics. 2025 Jun 11;24(7):101016. doi: 10.1016/j.mcpro.2025.101016.
Small open reading frame-encoded peptides (SEPs), translated from previously unannotated genomic regions, have emerged as important regulators in diverse physiological and pathological processes. While ribosome profiling and bioinformatics analysis can predict putative SEPs, mass spectrometry (MS) is the only method for their definitive identification. However, MS-based SEP detection faces significant challenges due to SEP's short length and low abundance. To address these limitations, we developed an ammonium formate-mediated C8 solid-phase enrichment (AmF-C8-SPE) strategy that significantly outperforms classic C8-SPE, yielding superior SEP identification with enhanced unique peptide ratios and sequence coverage. By coupling AmF-C8-SPE with fractionation and LC-MS/MS analysis of glioma samples from 18 patients, we identified 549 novel SEPs, 113 of which exhibited differential expression between tumors and adjacent normal tissues. Importantly, randomly selected SEPs were validated by MS spectral matching with synthetic peptides and by confirming recombinant fusion protein expression in cells. Furthermore, Mfuzz clustering and ROC curve analyses revealed SEPs associated with glioma progression. DeepLoc-based prediction followed by confocal microscopy imaging confirmed nuclear localization of two candidate SEPs (IP_613981 and SPROHSA206836). Therefore, this study establishes an optimized SEP identification approach and the first comprehensive SEP profiling in glioma, providing a valuable resource to discover novel glioma biomarker and therapeutic target.
从小的开放阅读框编码肽（SEPs），由先前未注释的基因组区域翻译而来，已成为多种生理和病理过程中的重要调节因子。虽然核糖体分析和生物信息学分析可以预测潜在的SEPs，但质谱（MS）是其确定性鉴定的唯一方法。然而，基于MS的SEP检测由于SEP的长度短和丰度低而面临重大挑战。为了解决这些限制，我们开发了一种甲酸铵介导的C8固相富集（AmF-C8-SPE）策略，该策略明显优于经典的C8-SPE，在提高独特肽比率和序列覆盖率的情况下产生了卓越的SEP鉴定结果。通过将AmF-C8-SPE与来自18例患者的胶质瘤样本的分级分离和LC-MS/MS分析相结合，我们鉴定出549种新型SEPs，其中113种在肿瘤组织和相邻正常组织之间表现出差异表达。重要的是，通过与合成肽的MS光谱匹配以及通过确认细胞中的重组融合蛋白表达，对随机选择的SEPs进行了验证。此外，Mfuzz聚类和ROC曲线分析揭示了与胶质瘤进展相关的SEPs。基于DeepLoc的预测随后通过共聚焦显微镜成像证实了两种候选SEPs（IP_613981和SPROHSA206836）的核定位。因此，本研究建立了一种优化的SEP鉴定方法以及胶质瘤中的首个全面SEP图谱，为发现新型胶质瘤生物标志物和治疗靶点提供了宝贵资源。
