Volume 35, No 3, Mar 2025

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 35 Issue 3, March 2025: 186-204   |  Open Access

ORIGINAL ARTICLES

Comprehensive discovery and functional characterization of the noncanonical proteome

Chengyu Shi^1,2,3,4,† , Fangzhou Liu^1,2,3,4,† , Xinwan Su^1,2,3,4,† , Zuozhen Yang^2,3,4 , Ying Wang^2,3,4 , Shanshan Xie^3,5,6 , Shaofang Xie⁷ , Qiang Sun¹ , Yu Chen^2,3,4 , Lingjie Sang^2,3,4 , Manman Tan^2,3,4 , Linyu Zhu^2,3,4 , Kai Lei^2,3,4 , Junhong Li^2,3,4 , Jiecheng Yang^2,3,4 , Zerui Gao^2,3,4 , Meng Yu^2,3,4 , Xinyi Wang^2,3,4 , Junfeng Wang^2,3,4 , Jing Chen⁸ , Wei Zhuo^3,5,6 , Zhaoyuan Fang^9,10 , Jian Liu^9,11 , Qingfeng Yan² , Dante Neculai¹ , Qiming Sun¹ , Jianzhong Shao² , Weiqiang Lin¹² , Wei Liu¹ , Jian Chen^3,8 , Liangjing Wang⁶ , Yang Liu¹³ , Xu Li⁷ , Tianhua Zhou^1,3,5,14,* , Aifu Lin^{1,2,3,4,15,16,*}

¹The Center for RNA Medicine, International Institutes of Medicine, International School of Medicine, The 4th Affiliated Hospital of Zhejiang University School of Medicine, Yiwu, Zhejiang, China
²MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China
³Cancer Center, Zhejiang University, Hangzhou, Zhejiang, China
⁴Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Hangzhou, Zhejiang, China
⁵Department of Cell Biology and Program in Molecular Cell Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
⁶Department of Gastroenterology, the Second Affiliated Hospital, School of Medicine and Institute of Gastroenterology, Zhejiang University, Hangzhou, Zhejiang, China
⁷Key Laboratory of Structural Biology of Zhejiang Province, Westlake Laboratory of Life Sciences and Biomedicine, Westlake University, Hangzhou, Zhejiang, China
⁸Department of Gastrointestinal Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
⁹Zhejiang University-University of Edinburgh Institute, Zhejiang University School of Medicine, Haining, Zhejiang, China
¹⁰The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
¹¹Hangzhou Cancer Hospital, Hangzhou, Zhejiang, China
¹²Department of Nephrology, Center for Regeneration and Aging Medicine, The Fourth Affiliated Hospital of School of Medicine and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, Zhejiang, China
¹³Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
¹⁴Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
¹⁵Future Health Laboratory, Innovation Center of Yangtze River Delta, Zhejiang University, Jiashan, Zhejiang, China
¹⁶Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Hangzhou, Zhejiang, China
^†These authors contributed equally: Chengyu Shi, Fangzhou Liu, Xinwan Su
Correspondence: Tianhua Zhou(tzhou@zju.edu.cn)Aifu Lin(linaifu@zju.edu.cn)

The systematic identification and functional characterization of noncanonical translation products, such as novel peptides, will facilitate the understanding of the human genome and provide new insights into cell biology. Here, we constructed a high-coverage peptide sequencing reference library with 11,668,944 open reading frames and employed an ultrafiltration tandem mass spectrometry assay to identify novel peptides. Through these methods, we discovered 8945 previously unannotated peptides from normal gastric tissues, gastric cancer tissues and cell lines, nearly half of which were derived from noncoding RNAs. Moreover, our CRISPR screening revealed that 1161 peptides are involved in tumor cell proliferation. The presence and physiological function of a subset of these peptides, selected based on screening scores, amino acid length, and various indicators, were verified through Flag-knockin and multiple other methods. To further characterize the potential regulatory mechanisms involved, we constructed a framework based on artificial intelligence structure prediction and peptide‒protein interaction network analysis for the top 100 candidates and revealed that these cancer-related peptides have diverse subcellular locations and participate in organelle-specific processes. Further investigation verified the interacting partners of pep1-nc-OLMALINC, pep5-nc-TRHDE-AS1, pep-nc-ZNF436-AS1 and pep2-nc-AC027045.3, and the functions of these peptides in mitochondrial complex assembly, energy metabolism, and cholesterol metabolism, respectively. We showed that pep5-nc-TRHDE-AS1 and pep2-nc-AC027045.3 had substantial impacts on tumor growth in xenograft models. Furthermore, the dysregulation of these four peptides is closely correlated with clinical prognosis. Taken together, our study provides a comprehensive characterization of the noncanonical proteome, and highlights critical roles of these previously unannotated peptides in cancer biology.

https://doi.org/10.1038/s41422-024-01059-3

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