*2.4. Precipitation-Based Isolation*

Polymer precipitation-based isolation has the benefits of commercial availability and easy processing and is now widely applied to isolate EVs from the biofluids under many disease statuses, such as colorectal cancer, epithelial ovarian cancer, and rheumatoid arthritis [67–69]. This type of isolation method is initially used in viral studies by forming a polymer network to decrease the

solubility of all components present in the sample [70]. The whole procedure includes mixing an appropriate volume of a polymer solution with samples and incubation. Then, the precipitated EVs are recovered by low-speed centrifugation. The polymer solution could be from a commercial kit, such as ExoQuick, Total Exosome Isolation, and ExoSpin, or home-made polyethylene glycol (PEG) solution [14]. Comparative studies have been conducted to evaluate the EVs isolated by different commercial kits in order to facilitate the choice of isolation methods. Ding et al. compared three commonly used commercial kits for EV isolation, including Total Exosome Isolation, ExoQuick, and RIBO Exosome Isolation Reagent. They found that the size of the majority of particles isolated by those kits was from 30–150 nm, while RIBO generated the highest particle yields. Further western blot (WB) results revealed that ExoQuick was the most efficient method by evaluating the marker proteins of CD63 and TSG101 [71]. Lobb et al. found that ExoSpin performed significantly better in avoiding co-isolation of contaminating proteins and yielded higher levels of EV markers compared to ExoQuick [55].

Although easy–to–use EV commercial kits are now widely used, home-made PEG has relative low-cost of EV preparation. Weng et al. added PEG into samples with a final concentration of 10% and incubated the samples at 4 ◦C for 2 h before recovery at centrifugation of 3000× *g*. Then a second-round of PEG precipitation was followed in order to improve the purity of EVs. The downstream MS analysis identified a total of 6299 protein groups from HeLa cell culture supernatant. Unfortunately, they did not test any biofluid sample in the study [72]. PEG has also been demonstrated to be used together with ultracentrifugation. Rider et al. purified the EVs resulted from one-round of PEG precipitation by further centrifugation at 100,000× *g* for 70 min [73]. Instead of isolating EVs by precipitation, aqueous two-phase systems (ATPSs) were proposed by Shin et al. They used a PEG/dextran ATPS to isolate EVs from the tumor interstitial fluid based on the mechanism that different kinds of particles are effectively partitioned to different phases in a short time. Their comparative studies showed that ATPSs could recovery about 70% of EVs from the EV protein mixtures, whereas the recovery for dUC and ExoQuick were about 16% and 40% [74]. But one should notice that EVs isolated by precipitation may be contaminated by polymer molecules, such as PEG, which is well-known for interfering in MS-based proteomic analysis. Therefore, it is necessary to remove those polymer molecules before MS analysis.
