Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells
Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on t...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-05-01
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| Series: | Redox Biology |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2213231723000435 |
| _version_ | 1811154754640805888 |
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| author | Ting Wu Shang-Tong Li Yu Ran Yinuo Lin Lu Liu Xiajun Zhang Lianqi Zhou Long Zhang Donghai Wu Bing Yang Shibing Tang |
| author_facet | Ting Wu Shang-Tong Li Yu Ran Yinuo Lin Lu Liu Xiajun Zhang Lianqi Zhou Long Zhang Donghai Wu Bing Yang Shibing Tang |
| author_sort | Ting Wu |
| collection | DOAJ |
| description | Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes. |
| first_indexed | 2024-04-10T04:22:23Z |
| format | Article |
| id | doaj.art-ffce4e1f07e34a6c8389cb6fee95b2f5 |
| institution | Directory Open Access Journal |
| issn | 2213-2317 |
| language | English |
| last_indexed | 2024-04-10T04:22:23Z |
| publishDate | 2023-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Redox Biology |
| spelling | doaj.art-ffce4e1f07e34a6c8389cb6fee95b2f52023-03-11T04:19:39ZengElsevierRedox Biology2213-23172023-05-0161102642Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cellsTing Wu0Shang-Tong Li1Yu Ran2Yinuo Lin3Lu Liu4Xiajun Zhang5Lianqi Zhou6Long Zhang7Donghai Wu8Bing Yang9Shibing Tang10Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Cancer Center, Zhejiang University, Hangzhou, 310058, ChinaGlbizzia Biosciences Co., Ltd, Beijing, 102601, ChinaZhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Cancer Center, Zhejiang University, Hangzhou, 310058, ChinaCenter for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China; China-New Zealand Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, ChinaZhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Cancer Center, Zhejiang University, Hangzhou, 310058, ChinaZhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Cancer Center, Zhejiang University, Hangzhou, 310058, ChinaZhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Cancer Center, Zhejiang University, Hangzhou, 310058, ChinaZhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Cancer Center, Zhejiang University, Hangzhou, 310058, ChinaCenter for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China; China-New Zealand Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, ChinaZhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China; Cancer Center, Zhejiang University, Hangzhou, 310058, China; Corresponding author. Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China.Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China; China-New Zealand Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China; Corresponding author. Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.http://www.sciencedirect.com/science/article/pii/S2213231723000435 |
| spellingShingle | Ting Wu Shang-Tong Li Yu Ran Yinuo Lin Lu Liu Xiajun Zhang Lianqi Zhou Long Zhang Donghai Wu Bing Yang Shibing Tang Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells Redox Biology |
| title | Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells |
| title_full | Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells |
| title_fullStr | Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells |
| title_full_unstemmed | Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells |
| title_short | Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells |
| title_sort | mapping protein direct interactome of oxidoreductases with small molecular chemical cross linkers in live cells |
| url | http://www.sciencedirect.com/science/article/pii/S2213231723000435 |
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