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Ayako Kurimoto
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P1.02 - Biology/Pathology (ID 614)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.02-064 - Proteomic Analysis of Exosome Purified Using a Novel Reagent (ID 10363)
09:30 - 16:00 | Presenting Author(s): Ayako Kurimoto
- Abstract
Background:
Exosomes are a type of extracellular vesicles secreted from all types of cells via endosomal pathway and found in most body fluids, including blood, urine, saliva, blood, breast milk, and cerebrospinal fluid. Many biologically active molecules such as protein, mRNA, miRNA, DNA and phospholipid are found in exosomes. Exosomes have been suggested to mediate cell-to-cell communication and breast cancer metastasis to lung via integrin of exosome. In order to explore the function of exosomes, highly efficient, comprehensive proteomic analysis is essential. To this end, surfactants are generally used to enhance protein digestion efficiency, which results in the increased total sequence coverage and number of identified peptides and proteins in LC-MS. In this study, we compared the efficiency of commercially available surfactants using cancer cell conditioned medium. We have also assessed the presence of cancer marker within the exosomes.
Method:
A novel reagent was added to serum, plasma, urine and conditioned medium, and exosomes were recovered by ultracentrifugation or immunoprecipitation. Obtained highly pure exosomes were subjected to immunoblotting, nanoparticle tracking analysis (NTA) and proteomic analysis using mass spectrometry. Obtained exosomes are collected from conditioned medium by ultracentrifugation, and lysed using commercially available MS-compatible detergents (e.g., RapiGest SF, AALS, NALS and ZALS) before being digested by proteases. Obtained peptides were analyzed using LC-MS.
Result:
LC-MS analysis has shown that the number of proteins identified from exosomes collected from the conditioned medium has increased by the addition of lysis step using various kinds of MS-compatible detergents compared to guanidine-HCl treatment, with the exception of CALS I and ZALS I.
Conclusion:
Highly pure exosomes can be isolated from cell culture supernatants and body fluids at high yield using our solution in immunoprecipitation and ultracentrifugation. Addition of solubilization step using detergents for proteomic analysis has increased the number of identified proteins from exosomes. However, the lysis efficiencies of exosomes varied depend on the type of surfactants.
