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. After removal of cells and cell debris, a speed of 110,000×g or 40,000×g was used for the ultracentrifugation or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the BCA assay, 1-D gel separation, transmission electron microscopy, Western blotting, and high resolution LC-MS/MS analysis. It was found that a five cycle repetition of ultracentrifugation or centrifugation is necessary for successful removal of non-exosomal proteins in the enrichment of exosomes from human serum. More significantly, 5×centrifugation enrichment was found to provide similar or better performance than 5×ultracentrifugation 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 CD proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5×ultracentrifugations 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.

Quantitative Proteomic Analysis of Serum Exosomes from Patients with Locally Advanced Pancreatic Cancer Undergoing Chemoradiotherapy

“…Five cycles of ultracentrifugation were necessary to purify exosomes and remove serum proteins including albumin. 27 The exosomes were suspended in 4 mL of PBS and centrifuged at 100 000 g for 60 min at 4 °C to clean the exosomes. This cleanup step was repeated three additional times.…”

Involvement of serum‐derived exosomes of elderly patients with bone loss in failure of bone remodeling via alteration of exosomal bone‐related proteins

“…For ultracentrifugation, the diluted serum was centrifuged at 10,000 ×  g for 30 min at 4°C followed by ultracentrifugation at 110,000 ×  g at 4°C for 90 min using a Beckman Optima L‐100XP Ultracentrifuge. The pellet was washed with 1 ml PBS and then dissolved with 40 μl 8 M urea (Kim, Tan & Lubman, 2015). The protein concentration was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific).…”

Protein content and functional characteristics of serum‐purified exosomes from patients with colorectal cancer revealed by quantitative proteomics

“…After the UC step, the supernatant was removed, followed by addition of 1 mL PBS buffer to wash the pellet without disturbing it. The pellet was then dissolved with 40 µL 8 M urea after the PBS was removed . The total protein concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific).…”