Hongbin Zhu, Betsy Benton, Chris Wojewodzki, Barbara Kaboord, and John C. Rogers,
Purpose: Comprehensive evaluation of six exosome isolation strategies using mass spectrometry-based proteomics and western blotting.
Methods: Exosomes were isolated from a pool of normal human serum using ultracentrifugation and five commercial exosome isolation kits: Exosomes were isolated, lysed using RIPA buffer or SDS, and the total protein was quantified using the BCA protein assay. A novel and rapid proteomic approach was performed for the profiling and quantification of exosome proteomes and biomarkers using a Thermo Scientific™ Q Exactive™ Plus platform. Biomarkers (CD9, CD81 CD63) and the contamination proteins were further validated by western blot. The proteome data were analyzed by Thermo Scientific™ Proteome Discoverer™ and the identified proteins were compared with the exosome database (ExoCarta) to evaluate the purity of the exosomes isolated using different strategies.
Results: We evaluated exosome isolation by ultracentrifugation and five commercial kits (Total Exosome Isolation Reagent for serum (Thermo Fisher Scientific), qEV iZON original (iZON Science), MagCapture Exosome Isolation PS (Wako Chemical), ExoEasy Maxi Kit (Qiagen), and Exo-spin™ Kits (Cell Guidance Systems)) that use different isolation methods. The initial evaluation comparing particle size and concentration showed that all methods yielded particles in the appropriate size range for exosomes (30-150nm). For proteomic profiling of different methods for exosome purification, the distribution of identified proteins was also compared. To assess the quality of the exosomes isolated from different strategies, we quantitatively monitored the enrichment of the identified exosome protein markers. In addition, western blotting was performed on each sample in parallel using known exosome surface markers. HSA was used as a negative control to assess purity. More proteins (620-700 protein groups) were identified from qEV iZon, MagCapture, UC_S (Ultracentrifugation with sucrose cushion purification) and ExoEasyMaxi than ExoSpin and Total Exosome Isolation Reagent (TEI). The results indicate that exosome purity and protein patterns are method dependent. MagCapture gives the best purity of exosomes for proteomics study among the six exosome isolation technologies. Higher purity of the isolated exosomes allows the identification of a higher number of proteins/exosome proteins. MagCapture gives the best exosome purity but the least protein yield (too low). Differential ultracentrifugation coupled with sucrose cushion does not provide the best exosome purity for proteomics study.
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