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A Modified Precipitation Method to Isolate Urinary Exosomes
Published on: January 16, 2015
A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes
Delfin A A Raj1, Immacolata Fiume, Giovambattista Capasso
1Laboratory of Mass Spectrometry and Proteomics, Institute of Protein Biochemistry - CNR, Naples, Italy.
Kidney International
|March 16, 2012
Summary
Researchers developed an improved ultracentrifugation method for isolating urinary exosomes, enabling efficient protein quantification for renal disease biomarker discovery. This method aids in identifying potential protein biomarkers for diagnosing kidney diseases.
Area of Science:
- Biochemistry
- Proteomics
- Renal Medicine
Background:
- Urinary exosomes are promising biomarkers for renal disease diagnosis.
- Current methods for exosome isolation, lysis, and protein quantification are inefficient.
- Efficient methods are crucial for advancing exosome-based protein biomarker research.
Purpose of the Study:
- To develop an improved ultracentrifugation-based method for urinary exosome isolation and protein analysis.
- To optimize exosome lysis, protein extraction, and digestion for quantitative proteomics.
- To identify potential protein biomarkers in urinary exosomes for renal disease diagnosis.
Main Methods:
- A two-step differential centrifugation followed by a double-cushion sucrose/D(2)O ultracentrifugation step was employed for exosome isolation.
- Uromodulin was removed to reduce impurities.
- Proteins were identified and quantified using MudPIT and four-plex iTRAQ analysis.
Main Results:
- The improved method effectively isolated urinary exosomes and removed major impurities.
- 378 and 79 unique proteins were identified in low- and high-density fractions, respectively.
- Six proteins (TSN1, PODXL, IDHC, PPAP, ACBP, and ANXA5) showed significant age-related expression differences in healthy men.
Conclusions:
- The developed ultracentrifugation method enhances urinary exosome isolation and protein solubilization.
- This method facilitates robust quantitative protein analysis of exosomes.
- The findings provide a foundation for developing reliable urinary exosome-based protein biomarker assays for renal diseases.
Related Concept Videos
01:36Overview of Exosomes
Exosomes are stable, lipid bilayer-enclosed vesicles capable of crossing biological barriers. They can carry a wide range of molecules required for intercellular communication. Once exosomes are released from the cell where they originated, they enter a recipient cell through various pathways such as fusion, receptor-mediated endocytosis, macropinocytosis, and phagocytosis.
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01:33Proteomics
A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term proteomics...
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