Editorial

Open AccessEditorial

Proteomic Analysis of Plasma-Derived Exosomes in Defining Their Role as Biomarkers of Disease Progression, Response to Therapy and Outcome

by

Theresa L. Whiteside

Proteomes 2019, 7(3), 27; https://doi.org/10.3390/proteomes7030027 - 30 Jun 2019

Cited by 10 | Viewed by 4011

Abstract

Extracellular vesicles (EVs) have recently emerged as an intercellular communication system that plays an important role in health and becomes dysfunctional in disease [...] Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

Research

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Open AccessFeature PaperArticle

Comparison of Proteome Composition of Serum Enriched in Extracellular Vesicles Isolated from Polycythemia Vera Patients and Healthy Controls

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Anna Fel

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Aleksandra E. Lewandowska

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Petro E. Petrides

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Jacek R. Wiśniewski

Proteomes 2019, 7(2), 20; https://doi.org/10.3390/proteomes7020020 - 06 May 2019

Cited by 19 | Viewed by 4669

Abstract

Extracellular vesicles (EVs), e.g., exosomes and microvesicles, are one of the main networks of intercellular communication. In myeloproliferative neoplasms, such as polycythemia vera (PV), excess of EVs originating from overabundant blood cells can directly contribute to thrombosis through their procoagulant activity. However, the [...] Read more.

Extracellular vesicles (EVs), e.g., exosomes and microvesicles, are one of the main networks of intercellular communication. In myeloproliferative neoplasms, such as polycythemia vera (PV), excess of EVs originating from overabundant blood cells can directly contribute to thrombosis through their procoagulant activity. However, the proteomic composition of these vesicles in PV patients has not been investigated before. In this work, we examined the proteomic composition of serum EVs of PV patients in comparison to healthy controls. We processed EV-enriched serum samples using the Multiple Enzyme Filter Aided Sample Preparation approach (MED-FASP), conducted LC-MS/MS measurements on a Q-Exactive HF-X mass spectrometer, and quantitatively analyzed the absolute concentrations of identified proteins by the Total Protein Approach (TPA). Thirty-eight proteins were present at statistically significant different concentrations between PV patients’ study group and healthy controls’ group. The main protein components deregulated in PV were primarily related to excessive amounts of cells, increased platelet activation, elevated immune and inflammatory response, and high concentrations of procoagulant and angiogenic agents. Our study provides the first quantitative analysis of the serum EVs’ proteome in PV patients. This new knowledge may contribute to a better understanding of the secondary systemic effects of PV disease and further development of diagnostic or therapeutic procedures. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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Open AccessFeature PaperArticle

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

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Proteomes 2019, 7(2), 18; https://doi.org/10.3390/proteomes7020018 - 28 Apr 2019

Cited by 59 | Viewed by 7593

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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Open AccessArticle

Isolation and Characterization of Small Extracellular Vesicles from Porcine Blood Plasma, Cerebrospinal Fluid, and Seminal Plasma

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Helena Kupcova Skalnikova

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Proteomes 2019, 7(2), 17; https://doi.org/10.3390/proteomes7020017 - 25 Apr 2019

Cited by 27 | Viewed by 6971

Abstract

Extracellular vesicles (EVs) are a highly attractive subject of biomedical research as possible carriers of nucleic acid and protein biomarkers. EVs released to body fluids enable indirect access to inner organs by so-called “liquid biopsies”. Obtaining a high-quality EV sample with minimum contaminants [...] Read more.

Extracellular vesicles (EVs) are a highly attractive subject of biomedical research as possible carriers of nucleic acid and protein biomarkers. EVs released to body fluids enable indirect access to inner organs by so-called “liquid biopsies”. Obtaining a high-quality EV sample with minimum contaminants is crucial for proteomic analyses using LC–MS/MS or other techniques. However, the EV content in various body fluids largely differs, which may hamper subsequent analyses. Here, we present a comparison of extracellular vesicle yields from blood plasma, cerebrospinal fluid, and seminal plasma using an experimental pig model. Pigs are widely used in biomedical research as large animal models with anatomy and physiology close to those of humans and enable studies (e.g., of the nervous system) that are unfeasible in humans. EVs were isolated from body fluids by differential centrifugation followed by ultracentrifugation. EVs were characterized according to protein yields and to the quality of the isolated vesicles (e.g., size distribution, morphology, positivity for exosome markers). In our experimental setting, substantial differences in EV amounts were identified among body fluids, with the seminal plasma being the richest EV source. The yields of pellet proteins from ultracentrifugation of 1 mL of porcine body fluids may help to estimate body fluid input volumes to obtain sufficient samples for subsequent proteomic analyses. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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Open AccessCommunication

Extracellular Vesicle Integrins Distinguish Unique Cancers

by

Stephanie N. Hurwitz

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David G. Meckes, Jr.

Proteomes 2019, 7(2), 14; https://doi.org/10.3390/proteomes7020014 - 11 Apr 2019

Cited by 37 | Viewed by 4674

Abstract

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote [...] Read more.

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in cancer EVs compared to vesicles secreted from benign epithelial cells. The total EV integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumor stage across a variety of epithelial cancer cells. In particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumor stage. Differential expression of integrins across cancer cells and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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Review

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Open AccessFeature PaperReview

Different Types of Cellular Stress Affect the Proteome Composition of Small Extracellular Vesicles: A Mini Review

by

Agata Abramowicz

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Piotr Widłak

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Monika Pietrowska

Proteomes 2019, 7(2), 23; https://doi.org/10.3390/proteomes7020023 - 23 May 2019

Cited by 23 | Viewed by 4389

Abstract

Extracellular vesicles (EVs) are well-known mediators of the cellular response to different stress factors, yet the exact mechanism of their action remains unclear. Hence, the characterization of their cargo, consisting of proteins, nucleic acids, and different classes of metabolites, helps to elucidate an [...] Read more.

Extracellular vesicles (EVs) are well-known mediators of the cellular response to different stress factors, yet the exact mechanism of their action remains unclear. Hence, the characterization of their cargo, consisting of proteins, nucleic acids, and different classes of metabolites, helps to elucidate an understanding of their function in stress-related communication. The unexpected diversity and complexity of these vesicles requires the incorporation of multiple technologically advanced approaches in EV-oriented studies. This mini review focuses on the invaluable role of proteomics, especially mass spectrometry-based tools, in the investigation of the role of small EVs in their response to stress. Though relatively few experimental works address this issue to date, the available data indicate that stress conditions would affect the composition of protein cargo of vesicles released by stressed cells, as evidenced by the functional importance of such changes in the context of the response of recipient cells. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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Open AccessReview

Challenges in the Isolation and Proteomic Analysis of Cancer Exosomes—Implications for Translational Research

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Proteomes 2019, 7(2), 22; https://doi.org/10.3390/proteomes7020022 - 15 May 2019

Cited by 18 | Viewed by 4939

Abstract

Exosomes belong to the group of extracellular vesicles (EVs) that derive from various cell populations and mediate intercellular communication in health and disease. Like hormones or cytokines, exosomes released by cells can play a potent role in the communication between the cell of [...] Read more.

Exosomes belong to the group of extracellular vesicles (EVs) that derive from various cell populations and mediate intercellular communication in health and disease. Like hormones or cytokines, exosomes released by cells can play a potent role in the communication between the cell of origin and distant cells in the body to maintain homeostatic or pathological processes, including tumorigenesis. The nucleic acids, and lipid and protein cargo present in the exosomes are involved in a myriad of carcinogenic processes, including cell proliferation, tumor angiogenesis, immunomodulation, and metastasis formation. The ability of exosomal proteins to mediate direct functions by interaction with other cells qualifies them as tumor-specific biomarkers and targeted therapeutic approaches. However, the heterogeneity of plasma-derived exosomes consistent of (a) exosomes derived from all kinds of body cells, including cancer cells and (b) contamination of exosome preparation with other extracellular vesicles, such as apoptotic bodies, makes it challenging to obtain solid proteomics data for downstream clinical application. In this manuscript, we review these challenges beginning with the choice of different isolation methods, through the evaluation of obtained exosomes and limitations in the process of proteome analysis of cancer-derived exosomes to identify novel protein targets with functional impact in the context of translational oncology. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

Open AccessReview

Melanoma-Derived Extracellular Vesicles: Focus on Their Proteome

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Magdalena Surman

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Ewa Stępień

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Małgorzata Przybyło

Proteomes 2019, 7(2), 21; https://doi.org/10.3390/proteomes7020021 - 13 May 2019

Cited by 20 | Viewed by 5226

Abstract

Malignant melanoma is one of the most aggressive types of cancer, and its incidence is increasing rapidly each year. Despite the extensive research into improved diagnostic and treatment methods, early detection and disease constraint still present significant challenges. As successful isolation protocols have [...] Read more.

Malignant melanoma is one of the most aggressive types of cancer, and its incidence is increasing rapidly each year. Despite the extensive research into improved diagnostic and treatment methods, early detection and disease constraint still present significant challenges. As successful isolation protocols have been developed, extracellular vesicles (EVs) have become the subject of extensive investigation in terms of their role in cancer progression and as a possible source of disease biomarkers. Besides functional studies, quantitative and qualitative proteomics have recently emerged as promising tools for the advancement of melanoma biomarkers. Nevertheless, the amount of data concerning the proteome of melanoma-derived EVs is still very limited. In this review we cover the current knowledge on protein content of melanoma-derived EVs, with a focus on their potential role in the development and progression of melanomas. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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