Super Enhanced Purification of Denatured-Refolded Ubiquitinated Proteins by ThUBD Revealed Ubiquitinome Dysfunction in Liver Fibrosis
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Le résumé fourni par la source
Ubiquitination is crucial for maintaining protein homeostasis and plays a vital role in diverse biological processes. Ubiquitinome profiling and quantification are of great scientific significance. Artificial ubiquitin-binding domains (UBDs) have been widely employed to capture ubiquitinated proteins. The success of this enrichment relies on recognizing native spatial structures of ubiquitin and ubiquitin chains by UBDs under native conditions. However, the use of native lysis conditions presents significant challenges, including insufficient protein extraction, heightened activity of deubiquitinating enzymes and proteasomes in removing the ubiquitin signal, and purification of a substantial number of contaminant proteins, all of which undermine the robustness and reproducibility of ubiquitinomics. In this study, we introduced a novel approach that combines denatured-refolded ubiquitinated sample preparation (DRUSP) with a tandem hybrid UBD for ubiquitinomic analysis. The samples were effectively extracted using strongly denatured buffers and subsequently refolded using filters. DRUSP yielded a significantly stronger ubiquitin signal, nearly three times greater than that of the Control method. Then, eight types of ubiquitin chains were quickly and accurately restored; therefore, they were recognized and enriched by tandem hybrid UBD with high efficiency and no biases. Compared with the Control method, DRUSP showed extremely high efficiency in enriching ubiquitinated proteins, improving overall ubiquitin signal enrichment by approximately 10-fold. Moreover, when combined with ubiquitin chain-specific UBDs, DRUSP had also been proven to be a versatile approach. This new method significantly enhanced the stability and reproducibility of ubiquitinomics research. Finally, DRUSP was successfully applied to deep ubiquitinome profiling of early mouse liver fibrosis with increased accuracy, revealing novel insights for liver fibrosis research.
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Le contrôle bibliographique ouvert
DOI retrouvé dans Crossref DOI retrouvé ; titre concordant.
- Titre Crossref
- Super Enhanced Purification of Denatured-Refolded Ubiquitinated Proteins by ThUBD Revealed Ubiquitinome Dysfunction in Liver Fibrosis
- Date Crossref
- 01/11/2024
- Éditeur
- Elsevier BV
- Type
- journal-article
Ce recoupement confirme des métadonnées liées au DOI. Il ne confirme ni la méthode ni les conclusions de l’étude, et il ne compte pas comme une seconde source scientifique indépendante.
Où se fait cette recherche
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Chinese Academy of Medical Sciences & Peking Union Medical College pays non établi dans la noticeUniversité ou école supérieure
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Beijing Proteome Research Center pays non établi dans la noticeStructure de recherche
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Hebei University pays non établi dans la noticeUniversité ou école supérieure
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Qingdao University State Key Laboratory of Medical Proteomics pays non établi dans la noticeUniversité ou école supérieure
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Affiliated Hospital of Qingdao University pays non établi dans la noticeÉtablissement de santé
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School of Basic Medical State Key Laboratory of Medical Proteomics pays non établi dans la noticeUniversité ou école supérieure
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State Key Laboratory of Medical Proteomics pays non établi dans la noticeStructure de recherche
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College of Chemistry and Materials Science pays non établi dans la noticeUniversité ou école supérieure
Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing Proteome Research Center et Hebei University, avec 5 autres affiliations.
Une affiliation ne permet pas de déduire la nationalité d’un auteur.