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

Sara Alameldin; Victor Costina; Hesham A Abdel-Baset; Katja Nitschke; Phillip Nuhn; Michael Neumaier; Maren Hedtke

doi:10.1016/j.plabm.2021.e00241

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

Pract Lab Med. 2021 Jun 23:26:e00241. doi: 10.1016/j.plabm.2021.e00241. eCollection 2021 Aug.

Authors

Sara Alameldin^{1

2}, Victor Costina¹, Hesham A Abdel-Baset², Katja Nitschke³, Phillip Nuhn³, Michael Neumaier¹, Maren Hedtke¹

Affiliations

¹ Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany.
² Clinical Pathology Department, Assiut University Hospital, Assiut, Egypt.
³ Department of Urology and Urosurgery, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany.

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.