Management of Tamm–Horsfall Protein for Reliable Urinary Analytics

Purpose Urinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm–Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted “contaminant” in urinary assays. Thus, th...

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Bibliographic Details
Published in:Proteomics. Clinical applications 2019-11, Vol.13 (6), p.e1900018-n/a
Main Authors: Xu, Xiaomeng, Barreiro, Karina, Musante, Luca, Kretz, Oliver, Lin, Hanfei, Zou, Hequn, Huber, Tobias B., Holthofer, Harry
Format: Article
Language:English
Subjects:
Adult
biomarker
Biomarkers
Biomarkers - urine
Contaminants
Design of experiments
Dialysis
Experimental design
Extracellular Vesicles - metabolism
Humans
Mass Spectrometry
Mathematical analysis
Microscopy, Electron, Transmission
Precipitates
Protein Denaturation
Proteins
Proteome - analysis
Proteomics
Proteomics - methods
Tamm–Horsfall protein
Transmission electron microscopy
Urea
Urea - chemistry
Ureas
urinary extracellular vesicles
Urine
Uromodulin - chemistry
Uromodulin - urine
Western blotting
Young Adult
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Summary:Purpose Urinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm–Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted “contaminant” in urinary assays. Thus, the aim is to develop a simple method to manage THP efficiently. Experimental design The uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of urea to optimize release of uEVs from sample. Presence of uEVs is confirmed by transmission electron microscopy, Western blotting, and proteomic profiling in MS. Results Using HFD with urea treatment for uEV isolation reduces sample complexity to a great extent. The novel simplified uEV isolation protocol allows comprehensive vesicle proteomics analysis and should be part of any urine analytics to release all sample constituents from THP trap. Conclusions and clinical relevance The method brings a quick and easy protocol for THP management during uEV isolation, providing major benefits for comprehensive sample analytics.
ISSN:1862-8346
1862-8354
1862-8354
DOI:10.1002/prca.201900018