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.

Keywords: AGO2, Argonaute protein; CSF, cerebrospinal fluid; EVs, extracellular vesicles; Exosomes; FDR, false discovery rate; Human plasma; ILVs, intraluminal vesicles; MVBs, multivesicular bodies; Mass spectrometry; SEC, size exclusion chromatography; Size-exclusion chromatography; UC, ultracentrifugation; Ultracentrifugation; cfNAs, Cell-free nucleic acids; ctDNA, circulating tumor DNA; miRNA, microRNA.

Fig. 1

(a) Coomassie blue staining of the second and third eluted fractions from SEC of 0.5 ml human plasma after SDS-PAGE. (b) Western blot for the exosomal marker CD9 for the same SEC fractions as in (a). We also used the exosomal marker CD63 after stripping the Nitrocellulose membrane with mild stripping buffer. (c) Exemplary Nanoparticle Tracking Analysis of fraction 2 of one sample after SEC.

Fig. 2

Workflow demonstrating the methodologies used in exosomes isolation and detection.

Fig. 3

Proteins enriched in exosomes were isolated by 2 methods; UC alone and UC coupled with SEC, and analysed by LC-MS. Proteins detected were compared using the ExoCarta database as a reference. Four different samples are shown.

Fig. 4

Protein quantification and analysis of contaminating plasma proteins (a) Diagram showing the difference in concentration of total protein after UC and SEC. (b). Table showing the percentage of residual peptides in some of the major contaminating plasma proteins when comparing isolating exosomes using ultracentrifugation (UC) alone or using UC coupled with size exclusion chromatography (SEC), using the mean from four different samples.(c)Table showing the percentage of residual contamination of 3 Albumin peptides, regarding signal intensity, when comparing isolating exosomes using ultracentrifugation coupled with size exclusion chromatography (SEC) compared to UC alone, using the mean from four different samples.