Comprehensive discovery and functional characterization of the noncanonical proteome

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.

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Publication Details

Journal
Cell Research
Published
2025-01-10
DOI
https://doi.org/10.1038/s41422-024-01059-3
Citations
12
Primary Topic
RNA and protein synthesis mechanisms
Type
article
Field-Weighted Citation Impact
9.67

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article

Comprehensive discovery and functional characterization of the noncanonical proteome

Zhaoyuan Fang, Manman Tan, Qingfeng Yan, Junfeng Wang et al.
12 citations
Cell Research
RNA and protein synthesis mechanisms
9.67
article

Comprehensive discovery and functional characterization of the noncanonical proteome

Zhaoyuan Fang, Manman Tan, Qingfeng Yan, Junfeng Wang, Qiang Sun, Wei Zhuo, Chengyu Shi, Jian‐Zhong Shao, Aifu Lin, Shanshan Xie, Lingjie Sang, Fangzhou Liu, Qiming Sun, Jiecheng Yang, Dante Neculai, Zerui Gao, Zuozhen Yang, Liangjing Wang, Tianhua Zhou, Weiqiang Lin, Xinwan Su, Jian Chen, Xu Li, Ying Wang, Yu Chen, Jian Liu, Wei Liu, Lei Kai, Junhong Li, Yu Meng, Xinyi Wang, Jing Chen, Linyu Zhu, Shaofang Xie, Yang Liu
article en
12 citations

Abstract

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.

Cell ResearchVol. 35(3)
Zhejiang International Studies University (CN), Ministry of Education of the People's Republic of China (CN), University of Toronto (CA), Westlake University (CN), Zhejiang Cancer Hospital (CN), Zhejiang Lab (CN), Second Affiliated Hospital of Zhejiang University (CN), Zhejiang University-University of Edinburgh Institute (CN), Hangzhou Cancer Hospital (CN), Zhejiang University of Technology (CN), Zhejiang University (CN)
National Natural Science Foundation of China, National Science and Technology Major Project, Natural Science Foundation for Young Scientists of Shanxi Province, Young Scientists Fund, Fundamental Research Funds for the Central Universities, National Science Fund for Distinguished Young Scholars, Medical Science and Technology Project of Zhejiang Province, Science and Technology Program of Zhejiang Province
Openalex Percentile: Top 2%
RNA and protein synthesis mechanisms
9.67
DOIOpen Access PDFOpenAlex
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