Citation: Tong Liu, Youdong Xu, Yajie Jiao, Jinguo Zhao, Bin Fu, Xianyu Li, Hongjun Yang, Weijie Qin. A dual-crosslinking and thiol-yne "click reaction"-based tagging method for mouse liver RNA binding proteome enrichment and identification by mass spectrometry[J]. Chinese Chemical Letters, ;2026, 37(1): 111154. doi: 10.1016/j.cclet.2025.111154 shu

A dual-crosslinking and thiol-yne "click reaction"-based tagging method for mouse liver RNA binding proteome enrichment and identification by mass spectrometry

Tong Liu ^{a, b, c} ,
Youdong Xu ^b ,
Yajie Jiao ^b ,
Jinguo Zhao ^{b, c} ,
Bin Fu ^b ,
Xianyu Li ^{a, *,} ,
Hongjun Yang ^{a, *,} ,
Weijie Qin ^{b, *,}

a.
Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing Key Laboratory of Traditional Chinese Medicine Basic Research on Prevention and Treatment for Major Diseases, Beijing 100700, China
b.
State Key Laboratory of Medical Proteomics, National Center for Protein Sciences (Beijing), Beijing 102206, China
c.
Beijing Proteome Research Center, Beijing 102206, China

phd_xianyuli@foxmail.com

hongjun0420@vip.sina.com

aunp_dna@126.com

Received Date: 4 September 2024
Revised Date: 7 February 2025
Accepted Date: 26 March 2025
Available Online: 27 March 2025

Figures(7)

RNA binding proteins (RBPs) are a crucial class of proteins that interact with RNA and play a key role in various biological process. Deficiencies or abnormalities of RBPs are closely linked to the occurrence and progression of numerous diseases, making RBPs potential therapeutic targets. However, the limited tissue penetration of 254 nm UV irradiation makes it difficult to efficiently crosslink weak and dynamic RNA–protein interactions in mammal tissues. Additionally, RNA degradation in metal catalyzed click reaction further hinders the enrichment of RNA-protein complexes (RPCs). Due to these inherent limitations, globally profiling the RNA binding proteome in mammal organs has long been a challenge. Herein, we proposed a novel method, which utilized a dual crosslinking with formaldehyde and 254 nm UV irradiation, metabolic labeling and metal-free thiol-yne click reaction to enable large-scale enrichment and identification of RBPs in mouse liver, called FTYc_UV. In this method, formaldehyde is first used to crosslink the crude RNA-protein complexes (cRPCs) in situ to address the problem of poor tissue penetration of 254 nm UV irradiation. Furthermore, this method integrates metabolic labeling with a metal-free thiol-yne click reaction to achieve non-destructive RNA tagging. After specifically RNA-RBPs crosslinking by 254 nm UV irradiation in tissue lysates, formaldehyde decrosslinking is employed to remove non-specific proteins, leading to effective enrichment of RPCs from mouse liver and thereby overcoming the poor specificity of formaldehyde crosslinking. Application of FTYc_UV in mouse liver successfully identified over 1600 RBPs covering approximately 75% of previously reported RBPs. Furthermore, 420 candidate RBPs, including 151 metabolic enzymes, were also obtained, demonstrating the sensitivity of FTYc_UV and the potential of this method for in-depth exploration of RNA–protein interactions in biological and clinical research.
- RNA binding proteins,
- Thiol-yne click reaction,
- Crosslinking,
- Enrichment,
- Mass-spectrometry
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