One-Step Enrichment and Quantitative Analysis of In Vivo Protein Complexes via Dimethylpiperidine Cross-Linker DPST
Jing Chen
Department of Chemical Physics, School of Chemistry and Materials Science, University of Science and Technology of China, 96 Jinzhai Road, Hefei, 230026 China
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Both authors contributed equally to this work.
Search for more papers by this authorDr. Hang Gao
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Both authors contributed equally to this work.
Search for more papers by this authorBowen Zhong
Department of Chemical Physics, School of Chemistry and Materials Science, University of Science and Technology of China, 96 Jinzhai Road, Hefei, 230026 China
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorAssoc. Prof. Zhou Gong
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, Hubei, 430071 China
Search for more papers by this authorAssoc. Prof. Chao Liu
School of Engineering Medicine & School of Biological Science and Medical Engineering, Beihang University, Beijing, 100191 China
Search for more papers by this authorAo Zhang
School of Engineering Medicine & School of Biological Science and Medical Engineering, Beihang University, Beijing, 100191 China
Search for more papers by this authorNan Zhao
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorYuwen Chen
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
University of Chinese Academy of Sciences, Beijing, 100039 China
Search for more papers by this authorBaofeng Zhao
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorProf. Zhen Liang
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorProf. Yukui Zhang
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorCorresponding Author
Prof. Lihua Zhang
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
E-mail: [email protected]; [email protected]
Search for more papers by this authorCorresponding Author
Prof. Qun Zhao
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
E-mail: [email protected]; [email protected]
Search for more papers by this authorJing Chen
Department of Chemical Physics, School of Chemistry and Materials Science, University of Science and Technology of China, 96 Jinzhai Road, Hefei, 230026 China
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Both authors contributed equally to this work.
Search for more papers by this authorDr. Hang Gao
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Both authors contributed equally to this work.
Search for more papers by this authorBowen Zhong
Department of Chemical Physics, School of Chemistry and Materials Science, University of Science and Technology of China, 96 Jinzhai Road, Hefei, 230026 China
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorAssoc. Prof. Zhou Gong
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, Hubei, 430071 China
Search for more papers by this authorAssoc. Prof. Chao Liu
School of Engineering Medicine & School of Biological Science and Medical Engineering, Beihang University, Beijing, 100191 China
Search for more papers by this authorAo Zhang
School of Engineering Medicine & School of Biological Science and Medical Engineering, Beihang University, Beijing, 100191 China
Search for more papers by this authorNan Zhao
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorYuwen Chen
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
University of Chinese Academy of Sciences, Beijing, 100039 China
Search for more papers by this authorBaofeng Zhao
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorProf. Zhen Liang
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorProf. Yukui Zhang
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
Search for more papers by this authorCorresponding Author
Prof. Lihua Zhang
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
E-mail: [email protected]; [email protected]
Search for more papers by this authorCorresponding Author
Prof. Qun Zhao
State Key Laboratory of Medical Proteomics National Chromatographic Research and Analysis Center CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 China
E-mail: [email protected]; [email protected]
Search for more papers by this authorAbstract
In vivo cross-linking mass spectrometry (XL-MS) enables the proteome-wide characterization of protein complexes in living cells. However, most XL-MS methods face significant sample loss during enrichment, limiting their applications to limited-quantity samples, and suffer from poor reproducibility (20%–40%), hindering precise quantification. To overcome these challenges, we developed a novel membrane-permeable cross-linker, 2,6-dimethylpiperidine disuccinimidyl tridecanoate (DPST), in which the dimethylpiperidinyl group enables one-step enrichment of cross-linked peptides via tandem mass tags (TMTs) antibody approach, eliminating sample loss from multi-step processes and allowing analysis from as few as 1E4 cells. DPST also allows the light and heavy isotopic labeling of cross-linked samples at the cellular level, which reduces inaccuracies from multi-step preparations. This generates reporter ions for precise MS2 quantification, improving the signal-to-noise ratio without increasing spectral complexity. Using DPST, we analyzed cross-links in primary neurons from single fetal mice and quantified the transient and weak interactions in dynamic liquid–liquid phase separation (LLPS) environments. Additionally, DPST's design supports multiple isotopic labeling configurations (e.g., 6-plex, 10-plex). Therefore, DPST provides a scalable and robust tool for in vivo XL-MS-based qualitative and quantitative analysis of living cells, even with limited sample quantities.
Conflict of Interests
The authors declare no conflict of interest.
Open Research
Data Availability Statement
The MS data were shared on iProX (https://www.iprox.org/), an official member of ProteomeXchange Consortium, and are publicly available as of the date of publication. iProX ID: IPX0010677000, ProteomeXchange ID: PXD059305.
Supporting Information
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