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Abstract: TH-PO969

Enrichment and Identification of Ubiquitinated Proteins in Urinary Extracellular Vesicles

Session Information

Category: Pathology and Lab Medicine

  • 1501 Pathology and Lab Medicine: Basic

Authors

  • Li, Yongqiang, Third Affiliated Hospital of Southern Medical University, Guangzhou, China
  • Zou, Hequn, Third Affiliated Hospital of Southern Medical University, Guangzhou, China
Background

Urinary extracellular vesicles(uEVs) is widely considered to represent pathological events in the kidneys and the urogenital epithelium. Ubiquitin mediates proteolysis through the enzymatic conjugation of UB to proteins that contain primary degradation signals. The aim of our current study was to establish a method to enrich and identify ubiquitinated protein in uEVs.

Methods

After 2000g centrifugation, supernatant 2000g was obtained from 1st morning urine. Then supernatant 2000g was processed by Hydrostatic dialysis filter using dialysis membrane with a molecular weight cutoff (MWCO) of 1,000 kDa. Protein-A Sepharose® IgG was used to concentrate ubiquitinated proteins , which is based on the binding of antibodies to Sepharose beads and subsequent cross-linked by Dimethyl pimelimidate(DMP). Coomassie staining, western blot and mass spectrographic analysis were used to characterize the ubiquitinated protein in uEVs.

Results

Ubiquitinated proteins in uEVs are enriched successfully. We detected 60 specific sites of 37 ubiquitinated proteins. These ubiquitinated proteins were mainly enriched in the biological process categories of cellular process. Most of the ubiquitinated proteins in the cellular component category occurred in the nucleus. The KEGG enrichment pathway analysis of these ubiquitinated proteins suggested that ubiquitination maybe play an indispensable role in the regulation of the mTor signaling pathway.

Conclusion

We provides a simplified method to enrich and identify ubiquitinated proteins in uEVs. 60 specific sites of 37 ubiquitinated proteins were discovered. Till now, all screening ubiquitinated proteins in urinary EVs of this study have not been previously reported. These method and findings allows the possibility to further chronic kidney disease studies in uEVs related to ubiquitinated proteins.

The proteins in red were identified in mTOR of this project

Funding

  • Government Support - Non-U.S.