Combination of Size-Exclusion Chromatography and Ultracentrifugation Improves the Proteomic Profiling of Plasma-Derived Small Extracellular Vesicles

Abstract

Background: Circulating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins and plasma proteins. Previous studies suggested combinations of different sEVs isolation methods could improve the yield and purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC), and their combination in a proteomic perspective. Results: Plasma samples were collected from healthy individuals, and sEVs were separated by one-step SEC, onestep UC, and combining SEC with UC, respectively. Here we exhibited that the purity of sEVs was improved by SEC in contrast to traditional UC. Furthermore, by conducting a SEC procedure followed by UC, we separated sEVs with the highest purity. In the proteomic analysis, 992 protein species were identified in the plasma sEVs isolated by our novel separation method, of which several proteins are sEVs-associated proteins but hitherto never been identified in the previous studies and database, much more than plasma sEVs isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, plasma sEVs isolated by the new procedure kept 584 previously identified sEVs-associated proteins and 360 other proteins that have not been detected before. Detailed analysis suggested that more kinds of sEVs biomarkers, such as CD9, ALIX, and FLOT1, could be identified in plasma sEVs isolated by the novel isolation method as compared to one-step UC/SEC. Furthermore, the lower abundance ranks of common contaminants, such as lipoproteins and IgG chains, in the sEVs fractions obtained by our new method as compared to one-step UC/SEC also demonstrated the purity of sEVs had been improved. Conclusions: Combining SEC with UC could significantly improve the performance of mass spectrometry-based proteomic profiling in analyzing plasma-derived sEVs.

Extracellular Vesicles and Exosomes: Insights From Exercise Science

“…Finally, given the restrictive process, SEC may also result in low vesicle yield, albeit with a greater purity compared to other methods. As with any developing methodology, a combination of methods (e.g., SEC followed by UC) will continue to be utilized and ultimately result in improvements over approaches that rely on single methods (Wei et al, 2020), though SEC-enriched ELVs remain an excellent methodological approach.…”

Radiolabelling of Extracellular Vesicles for PET and SPECT imaging

“…Previous studies have shown that sEVs isolated by ultracentrifugation can lead to co-precipitation of serum proteins (such as albumin) 120 , with lower purity as determined by the particle-to-protein ratio, compared to SEC 121 despite co-isolation of lipoproteins using this method 122 . Wei et al have shown that by combining these two methods, it is possible to improve elimination of contaminating proteins, lipoproteins and to improve particle-to-protein ratio 123 . Nevertheless, further evidence is needed to determine if this is the case for kidney/bladder/urine uptake of EVs.…”

A Novel Pool of Microparticle Cholesterol Is Elevated in Rheumatoid Arthritis but Not in Systemic Lupus Erythematosus Patients

“…The potential impact of microparticle preparations with cholesterol-rich lipoproteins must also be considered, and steps must be taken to ensure that microparticle preparations are essentially lipoprotein free. As the Svedberg coefficients of microparticles and lipoproteins overlap, it is extremely difficult to ensure that there is separation when using ultracentrifugation-based methods to isolate microparticles [23]. It is also difficult to interpret the microparticle lipid content in such studies where there may be contamination with plasma lipoproteins and their constituent proteins [5].…”

“…Studies of microparticle cholesterol have been hampered by difficulties in separating microparticles from cholesterol-rich lipoproteins which share similar biophysical properties [20]. However, using a combination of size-exclusion methods with ultracentrifugation has emerged in recent years which facilitate the isolation of lipoprotein-deficient microparticles [23]. In this paper we describe the application of these methods to characterise microparticle cholesterol in healthy controls, RA and SLE patients.…”