Exosome enrichment of human serum using multiple cycles of centrifugation

Abstract

In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high-resolution LC-MS/MS analysis. It was found that a five-cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD-63, and numbers of identified exosome-related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome-related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum.

Keywords: Centrifugation; Exosomes; Human serum; Mass spectrometry; Ultracentrifugation.

Figure 1

1-D gel images for the samples from (A) ultracentrifugation and (B) centrifugation processes of 2.0 mL human serum. The samples from 1 through 5 (U1–U5 and C1–C5) are the supernatants from the corresponding enrichment processes. The samples of U6 and C6 are from the enriched exosomes. The samples of 1 and 2 were diluted 500-fold and 20-fold with a PBS buffer solution prior to loading to reduce their concentrations and provide weaker bands.

Figure 2

Western blot analyses detecting CD-63 in exosomes purified from human serum using (A) the ultracentrifugation enrichment for 5 times and (B) the centrifugation enrichment for 5 times. The columns of “1”, “2”, and “3” for each image are from the exosome proteins obtained from 4 mL, 2 mL, and 1 mL human serum, respectively.

Figure 3

TEM images of exosome samples enriched from human serum using 5×ultracentrifugations and 5×centrifugations. The first, the second and the third rows show the images for the exosomes enriched from starting amounts of 1 mL, 2 mL, and 4 mL, respectively. Scale bars, 100 nm.

Figure 4

Histograms showing the diameter distribution of exosomes enriched from 4 mL human serum using (A) 5×ultracentrifugation enrichment and (B) 5×centrifugation enrichment. The total number of exosome particles used for each histogram is shown as “n”.

Figure 5

Venn diagram showing the overlap of exosome proteins enriched from 5×ultracentrifugations and 5×centrifugations. A total of 37 CD proteins were identified.

Figure 6

Venn diagram showing the overlap of exosome proteins enriched from the current investigation and from the three different enrichment methods [2]. UC, ultracentrifugation; C, centrifugation; DG, density gradient; EI, EpCAM-based immunoaffinity pull-down.

Scheme 1

Summary of the current investigation