Coupling size exclusion chromatography to ultracentrifugation improves detection of exosomal proteins from human plasma by LC-MS

Abstract

Objectives Exosomes are small lipid bilayer vesicles that are defined by their endocytic origin and size range of 30–140 nm. They are constantly produced by different cell types, by both healthy and abnormal cells, and can be isolated from almost all body fluids. Little information exists in isolating exosomes from plasma due to the complexity of its content and the presence of contaminating plasma proteins. Design and methods We carried-out liquid chromatography-mass spectrometry (LC-MS/MS) analyses of plasma-derived vesicles from 4 healthy donors obtained by 2 coupled methodologies: Ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC) to isolate and subsequently enrich exosomes. We compared the proteins detected by UC alone and UC coupled with SEC. Results In the coupled UC + SEC methodology we found 52.25% more proteins enriched in exosomes as CD9, Annexins, YWHAZ (14-3-3 family) and others, than by using UC alone. There is also a reduction of 98.8% of contaminating plasma proteins by coupling UC and SEC in comparison to using UC alone. Conclusions We conclude that exosomes can be successfully isolated from plasma using a very simple combination of standard methods, which could largely improve the proteomics profiling of plasma exosomes.

Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles

“…Here, we present a complete analysis of an optimised protocol for the isolation of sEVs from cells grown in serum‐containing media based on tangential flow filtration and subsequent size exclusion chromatography. The coupling of ultracentrifugation and size exclusion chromatography is a common technique employed by EV researchers (Alameldin et al., 2021 ; Koh et al., 2018 ; Stam et al., 2021 ). For this reason, it was important to comprehensively assess sEVs isolated by ultracentrifugation and tangential flow filtration, in conjunction with size exclusion chromatography.…”

Recent developments in isolating methods for exosomes

“…On this basis, many researchers have integrated and innovated on the existing methods. For example, size-exclusion chromatography combined with iodixanol density gradient centrifugation was used for isolation, aiming to achieve high yield and purity (Alameldin et al, 2021).…”

Biological Features of Extracellular Vesicles and Challenges

“…Indeed, EV purification techniques such as immunoaffinity capture, size exclusion, polymer precipitation, differential ultracentrifugation and microfluidic techniques are not always mutually exclusive, and their combined use may enhance the effectiveness of isolation and purification (Stam et al, 2021). Using ultra-high centrifugation to enrich exosomes, followed by a size-exclusion column could effectively remove soluble proteins and other contaminants (Koh et al, 2018), and also improve the proteomics profiling of plasma exosomes (Alameldin et al, 2021). Circulating tumor cells and exosomes could isolate by using microfluidic device with tumorspecific antibodies (Kang et al, 2020).…”