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

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.