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Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components

1
Maria Skłodowska-Curie Institute—Oncology Center, Gliwice Branch, 44-101 Gliwice, Poland
2
Department of Histology and Cell Pathology, School of Medicine with Division of Dentistry in Zabrze, Medical University of Silesia, 41-800 Zabrze, Poland
*
Author to whom correspondence should be addressed.
Proteomes 2019, 7(2), 18; https://doi.org/10.3390/proteomes7020018
Received: 26 March 2019 / Revised: 25 April 2019 / Accepted: 26 April 2019 / Published: 28 April 2019
(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)
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Abstract

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available. View Full-Text
Keywords: exosome; mass spectrometry; serum proteome; size-exclusion chromatography exosome; mass spectrometry; serum proteome; size-exclusion chromatography
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Smolarz, M.; Pietrowska, M.; Matysiak, N.; Mielańczyk, Ł.; Widłak, P. Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components. Proteomes 2019, 7, 18.

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