PROTEOMICS OF EXTRACELLULAR VESICLES: EXOSOMES AND ECTOSOMES Dong-Sic Choi, Dae-Kyum Kim, Yoon-Keun Kim, and Yong Song Gho* Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea Received 15 July 2013; revised 9 December 2013; accepted 9 December 2013 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21420

Almost all bacteria, archaea, and eukaryotic cells shed extracellular vesicles either constitutively or in a regulated manner. These nanosized membrane vesicles are spherical, bilayered proteolipids that harbor specific subsets of proteins, DNAs, RNAs, and lipids. Recent research has facilitated conceptual advancements in this emerging field that indicate that extracellular vesicles act as intercellular communicasomes by transferring signals to their target cell via surface ligands and delivering receptors and functional molecules. Recent progress in mass spectrometry-based proteomic analyses of mammalian extracellular vesicles derived from diverse cell types and body fluids has resulted in the identification of several thousand vesicular proteins that provide us with essential clues to the molecular mechanisms involved in vesicle cargo sorting and biogenesis. Furthermore, cell-type- or disease-specific vesicular proteins help us to understand the pathophysiological functions of extracellular vesicles and contribute to the discovery of diagnostic and therapeutic target proteins. This review focuses on the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles (i.e., exosomes and ectosomes), EVpedia (a free web-based integrated database of high-throughput data for systematic analyses of extracellular vesicles; http://evpedia.info), and the intravesicular protein– protein interaction network analyses of mammalian extracellular vesicles. The goal of this article is to encourage further studies to construct a comprehensive proteome database for extracellular vesicles that will help us to not only decode the biogenesis and cargo-sorting mechanisms during vesicle formation but also elucidate the pathophysiological roles of these complex extracellular organelles. # 2014 Wiley Periodicals, Inc. Mass Spec Rev Keywords: intercellular communicasomes; nanocosmos; intravesicular protein–protein interaction networks; proteomics; systems biology

Additional supporting information may be found in the online version of this article at the publisher’s web-site. Contract grant sponsor: National Research Foundation of Korea (NRF); Contract grant numbers: 2013035248, 2012R1A1A2042534; Contract grant sponsor: Ministry of Health and Welfare; Contract grant number: A120273; Contract grant sponsor: Korea Basic Science Institute; Contract grant number: D33400.
Correspondence to: Yong Song Gho, Department of Life Sciences, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 790-784, Republic of Korea. E-mail: [email protected]

Mass Spectrometry Reviews # 2014 by Wiley Periodicals, Inc.

I. INTRODUCTION Throughout the evolutionary process, bacteria, archaea, and eukaryotic cells have secreted nanosized membrane vesicles into the extracellular space (Fig. 1) (Lee et al., 2008, 2009; Deatherage & Cookson, 2012; Choi et al., 2013a). These extracellular vesicles are spherical, bilayered proteolipids that are enriched with various proteins, nucleic acids, and lipids. Although they have long been considered cellular artifacts or dust, recent progress in this area indicates that extracellular vesicles are intercellular communicasomes, that is, extracellular organelles that have multifaceted physiological and pathological functions in intercellular communication as well as inter-species and inter-kingdom communication (Choi et al., 2007, 2011; Lee et al., 2008, 2010; Hong et al., 2009). Furthermore, the secretion of extracellular vesicles could have similar features in different organisms, and extracellular vesicle-mediated intercellular communication is evolutionarily conserved (Lee et al., 2009; Deatherage & Cookson, 2012; Choi et al., 2013a). Publications regarding prokaryotic and eukaryotic extracellular vesicles have grown rapidly during the last 10 years (Fig. 2); this rapid growth indicates that the field of extracellular vesicles is expanding intensively (Kim et al., 2013). Therefore, the study of extracellular vesicles provides crucial clues for understanding the intercellular communication network in living organisms and the evolutionary connections among bacteria, archaea, and eukaryotes.

A. Overview of Mammalian Extracellular Vesicles: Exosomes and Ectosomes Extracellular vesicles are secreted by various mammalian cells under physiological conditions and various disease states, including cancer, in which the release of extracellular vesicles is aberrantly increased (Gyorgy et al., 2011). Two mechanisms of mammalian extracellular vesicle biogenesis have been suggested (Fig. 3A): (1) exosomes that are 50–100 nm in diameter are secreted from the endosomal membrane compartment after the fusion of multivesicular bodies (MVBs) with a plasma membrane or (2) ectosomes (also known as microparticles and microvesicles) with a diameter of 100 nm to 1 mm that are shed directly from the plasma membrane. These two types of mammalian extracellular vesicles harbor various bioactive materials including proteins, genetic materials (mRNAs and miRNAs), and lipids (Fig. 3B). The term exosomes is consistently used to indicate the exocytic vesicles from MVBs, whereas ectosomes are known by diverse names, including extracellular membrane vesicles, membrane particles, exovesicles, nanoparticles, microvesicles, microparticles, matrix vesicles, oncosomes, and shedding vesicles (Choi et al., 2013a).

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FIGURE 1. Secretion of extracellular vesicles and extracellular vesicle-mediated intercellular communication is evolutionarily conserved from prokaryotes (archaea, Gram-negative bacteria, and Gram-positive bacteria) to eukaryotes including mammalian cells.

target cells (Fig. 3A) using the following mechanisms (Ratajczak et al., 2006): (1) inducing receptor-mediated intracellular signal transduction by surface-expressed or bound ligands, (2) transferring surface receptors to target cells, and (3) delivering functional proteins, RNAs, and lipids into the target cells by fusion with plasma membrane or internalization into the endocytic compartment. These vesicle-mediated intercellular communications have several advantages. First, extracellular vesicles display the membrane proteins with a high local concentration. For example, vesicular ICAM1 has a lower mobility than soluble ICAM1, which likely increases the local density and overall avidity of ICAM1 (Lee et al., 2010). Second, extracellular vesicles have an endogenous ability to target their

Despite apparent differences in the mechanism of biogenesis between exosomes and ectosomes, it is experimentally difficult to discriminate exosomes from ectosomes after they are secreted from cells. Furthermore, there is no clearly descriptive physical property or molecular marker that can unambiguously distinguish exosomes from ectosomes (Deneka et al., 2007; Simons & Raposo, 2009; Choi et al., 2012b). Therefore, we refer to these mixed vesicle populations as extracellular vesicles (Fig. 3B) (Choi et al., 2012b). Upon release, both types of extracellular vesicles circulate in the local extracellular space and travel long distances by diffusion through body fluids, such as blood and lymph. Extracellular vesicles deliver complex signals via stimulating

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FIGURE 2. Publication trends on prokaryotic and eukaryotic extracellular vesicle studies. Publications on prokaryotic and eukaryotic extracellular vesicles that were manually curated after searching candidate papers in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) using the following keywords exosome
, exovesicle
, “membrane vesicle,” “membrane vesicles,” “extracellular vesicle,” “extracellular vesicles,” microvesicle
, argosome
, ectosome
, tolerosome
, “matrix vesicle,” “matrix vesicles,” oncosome
, dexosome
, “membrane particle,” “membrane particles,” prostasome
, nanovesicle
, “budding vesicle,” “budding vesicles,” “shedding vesicle,” “shedding vesicles,” “blebbing vesicle,” “blebbing vesicles,” “outer membrane bleb,” and “outer membrane blebs” (Kim et al., 2013).

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Ectosomes

Fusion

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Gelsolin Syntenin Calpain

Syndecan

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Sphingomyelin

Endocytosis

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Receptors:

CD9, CD63, CD81

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Cytoskeleton proteins: Actin, cofilins, ezrin/radixin/ moesin, myosin, tubulin

Lipids

Cytosolic proteins: 14-3-3 proteins, enolases, GAPDH, HSPs

Genetic materials : Ceramide : Cholesterol

: Phosphatidylserine : Sphingomyelin

mRNAs miRNAs

Vesicle traffickingrelated proteins: Annexins, ARFs, ALIX, ESCRTs, TSG101, RABs

Transporters and channels: Solute carrier family, ATP-binding cassettes

FIGURE 3. Two types of mammalian extracellular vesicles and their molecular components. A: Based on their biogenesis mechanisms, mammalian cells secrete two types of extracellular vesicles: exosomes and ectosomes. Exosomes, which are 50–100 nm in diameter, are secreted by the fusion of late endosomes or multivesicular bodies with the plasma membrane. Ectosomes (also known as microparticles and microvesicles), which are 100 nm to 1 mm in diameter, are shed directly from the plasma membrane. Released extracellular vesicles deliver complex signals to the target cells via ligand-receptor interaction, fusion with the plasma membrane, internalization by endocytosis, or unidentified mechanisms. B: Mammalian extracellular vesicles are enriched with cytoskeletal proteins, cytosolic proteins, secreted proteins, plasma membrane proteins (e.g., adhesion proteins, receptors, tetraspanins, and transporters), and vesicle trafficking-related proteins. Their membranes are enriched with cholesterol, ceramide, phosphatidylserine, and sphingolipids. Moreover, mammalian extracellular vesicles contain vesicle-specific genetic materials, such as DNAs, mRNAs, and miRNAs.

cells of origin. Third, extracellular vesicles can transfer functional hydrophobic molecules, such as bioactive lipids and membrane proteins. For example, vesicular sphingomyelin exhibits angiogenic activity by inducing the migration and proliferation of endothelial cells (Kim et al., 2002). In addition, extracellular vesicles promote the oncogenic transformation of target cells by transferring vesicular EGFRvIII (Al-Nedawi et al., 2008). Fourth, luminal components of extracellular vesicles are protected from degradation. For example, RNAs (mRNAs and miRNAs) are present in extracellular vesicles that are separated from body fluids, which are rich in RNase (Valadi et al., 2007).

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B. “Multi-Omics” Research on Extracellular Vesicles To decode the molecular mechanisms that are involved in vesicular cargo sorting and biogenesis as well as the diverse physiological and pathological functions of these complex extracellular organelles, several thousand protein and RNA components of extracellular vesicles that are derived from various cell types and body fluids have been catalogued using proteomic and transcriptomic methods (Choi et al., 2013a). However, relatively few vesicular lipids have been identified (Subra et al., 2007; Choi et al., 2013a). Systematic analyses of vesicular components have revealed that extracellular vesicles 3

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harbor a specific subset of proteins, genetic materials (mRNAs and miRNAs), and lipids rather than random cellular components (Fig. 3B). Moreover, these vesicular proteins, mRNAs, miRNAs, and lipids can be grouped as either common vesicular or cell-type-specific vesicular subsets. The common vesicular subset of proteins, mRNAs, miRNAs, and lipids provide us some essential clues regarding the molecular mechanisms that are involved in vesicle cargo sorting and biogenesis, whereas the cell-type-specific vesicular cargo help us to understand the pathophysiological functions of extracellular vesicles and could prove to be a rich source of biomarkers (Kosaka, Iguchi, & Ochiya, 2010; Raimondo et al., 2011; Choi et al., 2013a,b). This review focuses primarily on the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles (i.e., exosomes and ectosomes). A recent review of the proteomic analysis of Gram-negative bacteria extracellular vesicles (also known as outer membrane vesicles) has been published (Lee et al., 2008). In addition, we introduce EVpedia, which is a free web-based integrated database of high-throughput data for the systematic analyses of extracellular vesicles (http://evpedia.info), and we highlight the protein–protein interaction network analyses of mammalian extracellular vesicles that represent the interrelationship between vesicular proteins and their functional activities. The goal of this review is to encourage further studies on the high-throughput mass spectrometry-based proteomics and on the intravesicular protein–protein interaction network analyses of extracellular vesicles that provide us with essential clues to the molecular mechanisms involved in vesicle cargo sorting and biogenesis. Furthermore, cell-type- or disease-specific vesicular proteins help us to understand the pathophysiological functions of these complex extracellular organelles and contribute to the discovery of diagnostic and therapeutic target proteins.

II. BIOGENESIS OF MAMMALIAN EXTRACELLULAR VESICLES A. Exosomes Exosomes were first observed approximately 30 years ago by electron microscopy as small intracellular vesicles that contain select plasma membrane proteins in maturing reticulocytes (Pan et al., 1985). These small vesicles were segregated within a membrane-bound organelle forming a MVB and were released via fusion of the MVB’s external membrane with the plasma membrane of the cell (Fig. 3A). This process does not proceed by default or at random, but rather it is highly regulated by specific modulators. The endosomal sorting complexes required for transport (ESCRTs) have been shown to play a key role in the generation of vesicles in MVBs via the recognition of ubiquitinylated membrane proteins (Simons & Raposo, 2009). ESCRT complexes are composed of ESCRT-0, ESCRT-1, ESCRT-2, ESCRT-3, and other accessory proteins (Hurley & Hanson, 2010). ESCRT-0, ESCRT-1, and ESCRT-2 have ubiquitin-interacting modules that are necessary for the sequential sorting of cargo that is destined for the MVBs. ESCRT-0 plays a role in the clustering of ubiquitinylated cargo. ESCRT-1 and ESCRT-2 are responsible for membrane budding, and ESCRT-3 cleaves the budded membrane by fission. Recently, syndecan heparan sulfate proteoglycans and their cytoplasmic adaptor syntenin have been shown to interact with ALIX, an 4

ESCRT-3-binding protein, thus controlling the formation of exosomes (Baietti et al., 2012). However, an alternative pathway for exosome formation has been suggested in which the metabolic pathway for ceramide synthesis from sphingomyelin, which is catalyzed by sphingomyelinase, induces exosomal protein sorting into MVBs (Trajkovic et al., 2008). However, the exact molecular mechanisms that are involved in exosome biogenesis have not yet been elucidated.

B. Ectosomes The first description of ectosomes, also frequently referred to by other terms, such as microparticles or microvesicles, was reported in 1967 (Wolf, 1967). In human plasma, the fragments that were derived from platelets were identified, and they were shown to have shapes that resembled small membrane vesicles (Wolf, 1967). It has been suggested that ectosome shedding is preceded by the budding of protrusions on the plasma membrane followed by detachment by fission of the budding site (Fig. 3A) (Ratajczak et al., 2006; Cocucci, Racchetti, & Meldolesi, 2009). Importantly, the shedding rate of the ectosomes increases dramatically upon stimulation rather than upon release from resting cells. In particular, an increased level of intracellular Ca2þ induces a strong ectosome-shedding response in various cell types (Pilzer et al., 2005; Ratajczak et al., 2006; Moskovich & Fishelson, 2007). The intracellular Ca2þ concentration alters the asymmetric phospholipid distribution of the plasma membrane by activating scramblase and floppase, resulting in the exposure of phosphatidylserine and phosphatidylethanolamine in the outer leaflet of the plasma membrane (Pap et al., 2009). Moreover, the level of Ca2þ contributes to the degradation of the cytoskeleton via the activation of Ca2þ-dependent proteases, such as calpain and gelsolin, followed by ectosome release (Pap et al., 2009). Recently, it was reported that RhoA signaling leads to the downstream activation of a Rho-associated coiled-coilcontaining protein kinase that stimulates the generation of ectosomes to regulate the actin cytoskeleton via cofilin (Li et al., 2012). Moreover, the GTP-binding protein ARF6 modulates the release of protease-loaded ectosomes (Muralidharan-Chari et al., 2009a). By acting through phospholipase D and ERK, ARF6 activates myosin light chain kinase, and the subsequent phosphorylation of myosin light chain regulates the release of ectosomes from invasive cells (Muralidharan-Chari et al., 2009a,b). However, the mechanisms of ectosome biogenesis and the sorting of proteins into ectosomes are more obscure than the mechanisms of exosome biogenesis and the sorting of proteins into exosomes.

III. MASS SPECTROMETRY-BASED PROTEOMIC ANALYSES OF MAMMALIAN EXTRACELLULAR VESICLES Early mass spectrometry-based proteomic analyses of mammalian extracellular vesicles mainly focused on the identification of highly abundant vesicular proteins. In these studies, proteins from purified extracellular vesicles were separated using gel electrophoresis, and then, abundant proteins were analyzed by mass spectrometry (Thery et al., 1999, 2001; van Niel et al., 2001; Skokos et al., 2003). Using these approaches, some of the important vesicular marker proteins, such as tetraspanins, Mass Spectrometry Reviews DOI 10.1002/mas

PROTEOMICS OF EXTRACELLULAR VESICLES

et al., 2013). The most commonly used vesicle isolation method is ultracentrifugation (Thery et al., 2006; Choi et al., 2013a). After the pre-clearing of cells, cell debris, and large apoptotic bodies by differential centrifugation with or without 0.1–0.2 mm filtration, mammalian extracellular vesicles are pelleted by ultracentrifugation or sucrose cushion ultracentrifugation (e.g., 100,000–200,000g) (Hong et al., 2009). The vesicle isolation yield is adequate, but various different types of particles (e.g., lipoproteins and viruses), small apoptotic bodies, aggregates, protein oligomers, or proteins that are non-specifically bound to extracellular vesicles should be co-sedimented by this isolation method. Recent progress in high-throughput mass spectrometrybased proteomic analyses of extracellular vesicles indicates that buoyant density gradient ultracentrifugation is one of the best vesicle isolation methods to remove the above-mentioned contaminants that are caused by ultracentrifugation- or sucrose cushion ultracentrifugation-based vesicle isolation methods. Recently, several alternative vesicle isolation methods, including immunoaffinity beads, flow field-flow fractionation, and gel filtration, were introduced for efficient vesicle isolation (Kang et al., 2008; Dean et al., 2009; Looze et al., 2009; Mathivanan et al., 2010; Choi et al., 2013a). However, the technical standardization for challenging methods that are used to isolate extracellular vesicles is urgently needed because the lists of vesicular proteins identified by mass spectrometry-based proteomic analyses are largely dependent on the methods that are used for the isolation of extracellular vesicles. Recent reviews of this subject are available (Choi et al., 2013a; Witwer et al., 2013). Prior to high-throughput mass spectrometry-based proteomic analysis, dynamic range reduction of vesicular proteins or

14-3-3 proteins, actin, annexins, and heat shock proteins, were identified, but only a small number of vesicular proteins (approximately 10–30 proteins per study) were discovered. During the last 20 years, as protein separation and mass spectrometry technology have improved, several thousand proteins of extracellular vesicles from various cell types and body fluids, including serum, urine, saliva, ascites, and breast milk, have been catalogued by high-throughput mass spectrometry-based proteomic studies (Choi et al., 2013a; Kim et al., 2013). The lists of all vesicular proteins that have been identified by these studies have been deposited in the free webbased EVpedia (http://evpedia.info) database (Fig. 4) (Choi et al., 2013a; Kim et al., 2013). An overview of the current methodological approaches in the high-throughput mass spectrometry-based proteomic analyses of extracellular vesicles is provided in Figure 5. Although all of the steps described in this overall scheme have influenced the quality of the results, the following two methodologies are critical prerequisites for highthroughput mass spectrometry-based proteomic analyses: (1) efficient isolation of mammalian extracellular vesicles without any contamination by non-vesicular components and (2) fractionation of peptides or proteins prior to mass spectrometric analysis to reduce the vesicular proteome complexity. A variety of vesicle isolation methods, including ultracentrifugation, immunoaffinity beads, flow field-flow fractionation, and gel filtration, are used for the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles from cell culture medium or body fluids, including serum, urine, saliva, ascites, and breast milk (Fig. 5). The detailed vesicle isolation methods for high-throughput mass spectrometry-based proteomic analyses are listed in the EVpedia (http://evpedia.info) database (Choi et al., 2013a; Kim

Database: Vesicular protein, mRNA, miRNA, and lipid

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FIGURE 4. EVpedia: an integrated database of high-throughput data for the systematic analyses of prokaryotic and eukaryotic extracellular vesicles. A total of 230,937 vesicular proteins, mRNAs, miRNAs, and lipids of prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles from 190 high-throughput datasets are deposited in the free web-based database of EVpedia (http://evpedia.info) (Kim et al., 2013). For the systematic analyses of prokaryotic and eukaryotic extracellular vesicles, EVpedia provides an array of analytical tools: (1) search for and browse vesicular components, (2) Gene Ontology and network analyses of vesicular components, and (3) set analysis: a comparison of vesicular datasets by ortholog identification.

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FIGURE 5. Overview of methodological approaches in high-throughput mass spectrometry-based proteomic analyses of extracellular vesicles.

peptides by fractionation is critical to identify the highly confident vesicular proteins and to catalogue a large number of vesicular proteins (Choi et al., 2013a). As shown in Figure 5, three main strategies have been used to reduce the complexity of the vesicular proteome: (1) sodium dodecyl sulfate–polyacrylamide gel electrophoresis-based vesicular protein fractionation (Utleg et al., 2003; Pisitkun, Shen, & Knepper, 2004), (2) twodimensional liquid chromatography-based vesicular peptide fractionation, known as multidimensional protein identification technology (Gonzalez-Begne et al., 2009), and (3) isoelectric focusing-based fractionation of vesicular proteins or peptides (Choi et al., 2012a). In the following subsections, we summarize previous mass spectrometry-based proteomic studies of mammalian extracellular vesicles by introducing an integrated database of highthroughput data for the systematic analyses of extracellular vesicles (EVpedia) and discuss in detail the quantitation-based comparative proteomic analyses of mammalian extracellular 6

vesicles and the roles of proteomics in understanding mammalian extracellular vesicles.

A. EVpedia: An Integrated Database of HighThroughput Data for the Systematic Analyses of Extracellular Vesicles Currently, three public online databases of vesicular proteins, mRNAs, miRNAs, and lipids (EVpedia, ExoCarta, and Vesiclepedia) are available. EVpedia provides lists of the comprehensive vesicular proteins, mRNAs, miRNAs, and lipids of prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles (Choi et al., 2013a; Kim et al., 2013), while ExoCarta (Mathivanan & Simpson, 2009) and Vesiclepedia (Kalra et al., 2012) collects only the vesicular components of non-mammalian eukaryotic and mammalian extracellular vesicles. In the current EVpedia database (http://evpedia.info), a Mass Spectrometry Reviews DOI 10.1002/mas

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total of 230,937 vesicular proteins, mRNAs, miRNAs, and lipids from 190 high-throughput datasets were collected: 47,759 vesicular proteins deposited from the 97 high-throughput mass spectrometry-based proteomic datasets of mammalian extracellular vesicles (Fig. 4). Moreover, EVpedia provides an array of analytical tools: (1) search for and browse vesicular components, (2) Gene Ontology enrichment analysis of vesicular components, (3) network analysis of vesicular components, and (4) set analysis: a comparison of vesicular datasets by ortholog identification. In addition, the detailed methods for the isolation of extracellular vesicles and publications on extracellular vesicle studies are listed in the database. An overall comparison of EVpedia with Exocarta and Vesiclepedia has been recently published (Choi et al., 2013a; Kim et al., 2013). By providing these vesicle-specific components that are derived from various types of cells and body fluids as well as an array of analytical tools for systematic analyses, these integrated online databases facilitate communication and resource sharing between researchers in this emerging field of biology. Researchers can upload their high throughput datasets to the EVpedia database through the “Upload” menu (Kim et al., 2013).

Western blotting and two-dimensional gel electrophoresis (Lu et al., 2007). The currently available high-throughput quantitation-based comparative proteomic analyses of mammalian extracellular vesicles are summarized in Table 1. For example, we recently conducted APEX-based label-free quantitative proteomic analyses of extracellular vesicles derived from human primary colorectal cancer cells (SW480) and their metastatic derivatives (SW620) (Choi et al., 2012a). From the comparison of the estimated abundance of vesicular proteins, 368 and 359 enriched proteins were identified in SW480- and SW620-derived extracellular vesicles, respectively. These differentially regulated proteins are closely related to the cellular status. For example, SW620-enriched vesicular proteins are associated with cancer progression and multidrug resistance as potential diagnostic markers of metastatic cancer, whereas SW480-enriched vesicular proteins play a role in cell adhesion.

B. Quantitation-Based Comparative Proteomic Analyses of Mammalian Extracellular Vesicles

As more vesicular proteins are identified, it has become apparent that extracellular vesicles contain a specific subpopulation of proteins rather than randomly selected molecules from their cells of origin (de Gassart et al., 2004; Choi et al., 2013a). Vesicular proteins are mainly derived from the plasma membrane, cytosol, and internal vesicles and not from other intracellular organelles, such as the endoplasmic reticulum, Golgi apparatus, mitochondria, and nucleus. Moreover, extracellular vesicles harbor a common set of vesicular proteins and cell-type-specific components (Choi et al., 2013a). For example, annexins, cytoskeletal proteins (actins, cofilin-1, ezrin/radixin/ moesin, profilin-1, and tubulins), heat-shock proteins, integrins, metabolic enzymes (enolases, glyceraldehyde 3-phosphate dehydrogenase, peroxiredoxins, and pyruvate kinase), ribosomal proteins, tetraspanins (CD9, CD63, and CD81), and vesicle trafficking-related proteins (TSG101, ALIX, and RAB proteins, syntenin-1) are frequently identified vesicular proteins (Fig. 3B). Table 2 presents the top 100 vesicular proteins, ordered from the highest to lowest identification count, that are found most frequently in mammalian extracellular vesicles. The identification count of each vesicular protein represents the number of identifications of that protein in high-throughput proteomic datasets (Choi et al., 2013a). EVpedia (http://evpedia. info) provides a long list of vesicular proteins and their occurrences within prokaryotic and mammalian extracellular vesicles (Choi et al., 2013a; Kim et al., 2013). In addition, diverse functional proteins, including cell-surface antigens, immune-modulating proteins, proteases, and angiogenic molecules, have been identified (Choi et al., 2007). Therefore, these vesicular proteomes provide diverse information on the nature of extracellular vesicles and help us to decode the molecular mechanisms that are involved in vesicular cargo sorting and biogenesis as well as the diverse physiological and pathological functions of these complex extracellular organelles (Choi et al., 2007, 2013a). Finally, high-throughput mass spectrometrybased proteomic studies on extracellular vesicles facilitate biomarker discovery based on the protein signature of the originating cells. Recent reviews of this subject are available in the literature (Raimondo et al., 2011; Choi et al., 2013a,b).

Although most mammalian cells secrete extracellular vesicles, their release rates and their molecular components, including proteins, mRNAs, and miRNAs, are dependent on cell type. Furthermore, similar to the cellular proteome, vesicular protein components and their abundance are influenced by cellular status, including stimulation by growth factor (Di Vizio et al., 2009), exposure to the aging process (Lehmann et al., 2008), and transfection with infectious molecules (Coleman et al., 2012). Therefore, quantitation-based comparative proteomic analyses of mammalian extracellular vesicles that are derived from diverse cell types and cellular conditions are expected to provide insights into the emerging biology of extracellular vesicles, to elucidate the biogenesis and pathophysiological functions of extracellular vesicles and to contribute to the discovery of diagnostic and therapeutic target proteins. Two technical approaches are applied for protein quantitation in proteomics: label-based and label-free quantitation (Fig. 5). Label-based quantitation methods, such as isobaric tags for relative and absolute quantification (iTRAQ) and stableisotope labeling of amino acids (SILAC) are commonly used in various comparative proteomic analyses, but these laborious approaches are expensive, multistep labeling processes and are not always feasible due to the insufficient labeling of tags according to the discrimination of samples (Bachi & Bonaldi, 2008). Alternatively, label-free quantitation methods, based on the extracted chromatograms of spectral intensity or spectral counts, provide inexpensive, convenient, and reliable methods for quantitative proteomics studies (Bachi & Bonaldi, 2008). The spectral counting method is based on the assumption that more abundant proteins have more abundant peptides. The absolute protein expression (APEX) tool is based on spectral counting of each protein, and it is corrected by the prior expectation of observing each peptide (Lu et al., 2007). This approach provides absolute protein concentrations across approximately three to four orders of magnitude. Moreover, the protein abundance that is quantified by APEX is consistent with

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C. Role of Proteomics in Understanding Mammalian Extracellular Vesicles

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8

Unstimulated and stimulated by TNF-alpha and PAI-1 Exosomes and microparticles in patients of developmental heart defect Storage day 3, 21, and 42

Endothelial cell (HUVEC)

Deep venous thrombosis

Bladder cancer

Early IgA nephropathy and basement membrane nephropathy Bladder cancer and hernia

Plasma

Urine

Urine

−

−

−

−

+

−

−

+

−

+

+

+

+

+

+

+

−

−

−

+

Ultracentrifugation

−

+

Filtration (0.1-0.2 µm)

−

−

−

−

−

−

−

−

−

+

−

Sucrose cushion ultracentrifugation

−

+

−

−

+

−

−

−

−

+

−

Density gradient ultracentrifugation

−

+

+

−

−

+

+

−

+

+

−

Protein separation and in-gel digestion

+

−

−

+

−

−

−

−

−

−

+

In-solution digestion and peptide separation

Protein or peptide separation strategies

ESI-MS/MS

ESI-MS/MS

ESI-MS/MS

MALDIMS/MS

ESI-MS/MS

ESI-MS/MS

ESI-MS/MS

ESI-MS/MS

ESI-MS/MS

ESI-MS/MS

ESI-MS/MS

Mass spectrometer

Spectral intensity

Spectral counts Spectral intensity

iTRAQ

Spectral counts

Spectral counts

Spectral counts

Spectral counts

Total ion current

Spectral counts

Spectral counts

Protein Quantitation

2012

2011

2008

2010

2007

2011

2008

2012

2008

2012

2013

Year

Chen et al., 2012

Smalley et al., 2008 Moon et al., 2011

Ramacciotti et al., 2010

Admyre et al., 2007

Carayon et al., 2011

Bosman et al., 2008

Le Bihan et al., 2012

Peterson et al., 2008

Choi et al., 2012b

Beckler et al., 2013

Reference

ESI, electrospray ionization; iTRAQ, isobaric tags for relative and absolute quantification; LC, liquid chromatography; MALDI, matrix-assisted laser desorption; MS/MS, tandem mass spectrometry.

Urine

Colostrum and mature milk

Breast milk

Body fluid Homo sapiens

Reticulocyte

Rattus norvegicus

Red blood cell

Cultured for 36 h, 84 h, and 156 h

SW480 (Primary cancer) and SW620 (Metastatic cancer)

Colorectal cancer cell (SW480 and SW620)

Neonatal myoblast cell

DKs-8 (Wild-type KRAS), DLD-1 (Parental; heterozygous KRAS), and DKO-1 (Mutant KRAS)

Colorectal cancer cell (DKs-8, DLD1, and DKO-1)

Cell culture medium Homo sapiens

Sample type

Ultracentrifugation-based vesicle isolation

TABLE 1. High-throughput quantitation-based comparative proteomic analyses of mammalian extracellular vesicles

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TABLE 2. Top 100 vesicular proteins that are most often found in mammalian extracellular vesiclesa Protein name

Gene symbols

Identification count

1

Actin and related proteins

ACTB, ACTA2, ACTC1, ACTA1, ACTBL2, ACTG2

91

2

A n k yr i n

POTEF, POTEI, ACTG1, POTEKP, POTEE, POTEJ, POTEB

91

3

Heat shock protein 70

HSPA1A, HSPA5, HSPA8, HSPA6, HSPA2, HSPA1L, HSPA7

90

4

Ezrin/Radixin/Moesin

EZR, RDX, MSN

82

5

Glyceraldehyde-3-phosphate dehydrogenase/erythrose4-phosphate dehydrogenase

GAPDH, GAPDHS

78

6

Annexin A2

ANXA2, A NXA2 P2

76

7

L-lacta te de hydrog e na se

LDHA , L DHB , L DHAL6A

76

8

Peroxiredoxin

PRDX1, PRDX2, PRDX3

76

9

Eno las e

ENO1, ENO2 , EN O3

75

10

14-3-3 protein epsilon

YWHAE

72

11

Tubulin beta

TUBB, TUBB1, TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBB4B, TUBB6, TUBB8 CFL1

72

12

Cofilin-1

13

Heat shock protein 90

HSP90AA1, HSP90AB1, HSP90AB4P, TRAP1

HSP90B1,

HSP90AB3P,

71 70

14

Translation elongation factor EF-1alpha (GTPase)

EEF1A1, EEF1A2, EEF1A1P5, HBS1L

70

15

Pyruvate kinase

PKM, PKM2, PKLR

69

16

Tubulin alpha

68

17

3-phosphoglycerate kinase

TUBA4A, TUBA1C, TUBA8, TUBA3C, TUBA1A, TUBA1B, TUBA3E, TUBAL3 PGK1, PGK2

18

Guanine nucleotide-binding protein G(i) subunit alpha

G N A I 1 , G NA I 2, G N A I 3

67

19

Alpha-actinin

ACTN1, ACTN2, ACTN3, ACTN4

65

20

Ras-related protein Rab-5

RAB5B, RAB5C

65

21

14-3-3 protein zeta/delta

YWHAZ

64

22

Annexin A5

ANX A 5

64 64

67

23

Clathrin heavy chain

CLTCL1, CLTC

24

14-3-3 protein beta/alpha

YWHAB

63

25 26

Cation transport ATPase Guanine nucleotide-binding protein

ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP12A, ATP4A GNB1, GNB2, GNB4

63 63

27

Profilin-1

PF N1

63

28

Peptidyl-prolyl cis-trans isomerase

PPIA

61

29

Ras-related protein Rab-1A

RAB1A

60

30

Programmed cell death 6-interacting protein

PDCD6IP

59

31

14-3-3 protein theta

YWHAQ

58

32

14-3-3 protein gamma

YWHAG

57

33

Serum albumin

ALB

57 56

34

EH domain-containing protein

EHD1, EHD2, EHD3, EHD4

35

Ras-related protein Rap-1b

RAP1B

56

36

ADP-ribosylation factor 1

ARF1

55

37

Integrin beta

ITGB1, ITGB2, ITGB3, ITGB5, ITGB6, ITGB7

55

38

Peroxiredoxin-6

PRDX6

55

39

Glucose-6-phosphate isomerase

GPI

54

40

Syntenin-1

SDCBP

54

41

Triosephosphate isomerase

TPI1

54

42

Ubiquitin-like modifier-activating enzyme

UBA1, UBA6, UB A7

54

43

Annexin A6

ANXA6

53

44

Cell division control protein 42 homolog

CDC42

53

45

Fructose-1,6-bisphosphate aldolase

ALDOC, ALDOA

52

46

Translation elongation factors (GTPases)

EEF2, EFTUD2

52

47

CD9 antigen

CD9

51

48

Ras-related protein Rab-11

RAB11A, RAB11B

51

49

Annexin A7/11

ANXA7, ANXA11

50

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TABLE 2. (Continued) 50

Chloride intracellular channel protein 1

CLIC1

50

51

Fatty acid synthase

FASN

50

52

Myosin heavy chain

50

53

Ras-related protein Rab-7a

MYH1, MYH3, MYH4, MYH7, MYH8, MYH9, MYH10, MYH11, MYH14, MYH13, MYH15 RAB7A

54

4F2 cell-surface antigen heavy chain

SLC3A2

49

55

Eukaryotic initiation factor 4A

EIF4A1, EIF4A2

49

56

Ras-related C3 botulinum toxin substrate

RAC1, RAC2

49

57

T-complex protein 1 subunit beta

CCT2

49

58

Adenosylhomocysteinase

AHCY

48

59

ATP synthase subunit beta, mitochondrial

ATP5B

48

60

Complement

C3, C4A, C4B, C5

61

Myosin heavy chain 1

50

48

62

Rab GDP dissociation inhibitor

MYO1A, MYO1B, MYO1F, MYO1G GDI1, GDI2

MYO1C,

MYO1D,

MYO1E,

48

63

Keratin, type I cytoskeletal 10

KRT10

47

64

Large extracellular alpha-helical protein

A2M, A2ML1, CD109, PZP

47

48

65

Nucleoside diphosphate kinase

NME1, NME2, NME3

47

66

Phosphatidylethanolamine-binding protein 1

PEBP1

47

67

Ras-related protein Rab 1B/10

RAB1B, RAB10

47

68

Glutathione S-transferase P

GSTP1

46

69

Malate dehydrogenase

MDH1

46

70

Ras-related protein Ral-A

RALA

46

71

Transgelin-2

TAGLN2

46

72

Transitional endoplasmic reticulum ATPase

VCP

46

73 74

ACLY DST, FLNA, FLNB, FLNC, LCP1, PLEC, PLS1, PLS3

45 45

75

ATP-citrate synthase Ca2+-binding actin-bundling protein fimbrin/plastin (EF-Hand superfamily) Calcium ion binding protein

DSP, MACF1

45

76

Heat shock protein beta-1

HSPB1

45

77

Ras-related protein Rab-2B/14

R A B 1 4 , R A P 2B

45

78

Tyrosine rotein kinase

BLK, FGR, FYN, HCK, LCK, LYN, SRC, YES1

45

79

6-phosphogluconate dehydrogenase, decarboxylating

PGD

44

80

CD81 antigen

CD81

44

81

Clusterin

CLU

44

82

G e l s ol i n

GSN, SCIN, VIL1

44

83

Phosphoglycerate mutase

PGAM1, PGAM2

44

84

T-complex protein 1 subunit alpha

TCP1

44

85

Guanine nucleotide-binding protein G(q)

GNA11, GNAQ

43

86

Guanine nucleotide-binding protein subunit alpha-13

GNA13

43

87

Keratin, type II cytoskeletal 1

KRT1

43 43

88

Protein disulfide-isomerase A3

PDI A3

89

Ras-related protein Rab-2A

RAB2A

43

90

Talin

TLN1, TLN2

43

91

Erythrocyte band 7 integral membrane protein

STOM

42

92

G protein subunit G alphas

GNAL, G NAS, G NAS

42

93

Histocompatibility antigen

HLA-A, HLA-B, HLA-C, HLA-E, HLA-H

42

94

Ras-related protein Rab-8A

RAB8A

42

95

V-type proton ATPase catalytic subunit A

ATP6V1A

42

96

20S proteasome,alpha and beta subunits

PSMA7, PSMA8

41

97

SPTB, SPTBN1, SPTBN2, SPTBN4, SPTBN5

41

98

Ca2+-binding actin-bundling protein fimbrin/plastin (EF-Hand superfamily) Ca2+-binding protein (EF-Hand superfamily)

SPTAN1

41

99

Galectin-3-binding protein

LGALS3BP

41

100

Integrin alpha-2b/5

ITGA2B, ITGA5, ITGAV

41

EVpedia (http://evpedia.info) provides a large list of vesicular proteins and their occurrences of prokaryotic and mammalian extracellular vesicles (Choi et al., 2013a; Kim et al., 2013). a

The proteins are ordered from the highest to lowest identification count, which represents the number of identifications of that protein in highthroughput proteomic datasets (Choi et al., 2013a).

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IV. INTRAVESICULAR PROTEIN–PROTEIN INTERACTION NETWORKS OF MAMMALIAN EXTRACELLULAR VESICLES Although mass spectrometry-based proteomic analyses of mammalian extracellular vesicles have allowed several thousand vesicular proteins to be cataloged, their physical and functional interrelationships remain unclear. Our recent protein–protein interaction network analyses of mammalian extracellular vesicles indicate that vesicular proteins are physically and functionally interconnected to form functional modules involved in vesicle biogenesis and pathophysiological functions (Choi et al., 2012b, 2013a). Similar to protein–protein interaction networks of other intracellular organelles, including phagosomes, autophagosomes, and mitochondria, the overall topology of vesicular protein–protein interaction networks follows the power-law distribution of the existence of several hub proteins with other vesicular proteins having few interacting partners. This observation suggests that extracellular vesicles are nanocosmos (i.e., a nanosized orderly system of extracellular organelle), rather than cellular dust (Choi et al., 2012b; Kim et al., 2013). In this review, we expand our previous observations in extracellular vesicles that are derived from human colorectal cancer cells (SW480), human B cells, and human platelets, and we discuss the biological meaning of the protein–protein interaction between their vesicular proteins.

A. Intravesicular Protein–Protein Interaction Networks of Mammalian Extracellular Vesicles Using the previously reported vesicular proteomic data of SW480 (Choi et al., 2012a), B cell (Buschow et al., 2010), and platelet (Garcia et al., 2005), we first constructed intravesicular protein–protein interaction networks of mammalian extracellular vesicles (Fig. 6) (Choi et al., 2012b). Protein interaction data were gathered from BioGRID (version 3.2.101; http://www. thebiogrid.org/), which contains 130,292 physical interactions

SW480

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between 17,373 proteins. The protein–protein interaction networks were visualized using Cytoscape (Shannon et al., 2003). Similar to previous observations (Choi et al., 2012b, 2013a), vesicular proteins are interconnected via physical interactions, and their topologies follow the properties of scale-free networks by indicating several hub proteins with many other vesicular proteins having few interacting partners. These results suggest that physically interacting cellular proteins may be co-sorted into extracellular vesicles, as experimentally validated in our previous study (Choi et al., 2012b). Although the detailed molecular mechanisms by which proteins are loaded into extracellular vesicles during their biogenesis are not fully understood, increasing evidence indicates that the clustering, oligomerization, or protein–protein interaction among vesicular proteins play critical roles in vesicular cargo sorting (Vidal, Mangeat, & Hoekstra, 1997; Le Naour et al., 2006; Choi et al., 2012b). We previously reported that extracellular vesicles harbor functional modules that are comprised of physically interconnected tetraspanin web proteins (Choi et al., 2012b). The tetraspanin web is a well-known highly clustered protein complex in the cell (Le Naour et al., 2006). Moreover, the antibody-mediated cross-linking of plasma membrane proteins, such as the transferrin receptor in reticulocytes or the MHC II in lymphocytes, induces the sorting of these proteins into extracellular vesicles (Vidal, Mangeat, & Hoekstra, 1997).

B. Comparison of Extracellular Vesicles With Other Subcellular Compartments As shown in Figure 7, we next conducted analysis to identify biological processes enriched or depleted in extracellular vesicles relative to other intracellular organelles or compartments using a modified Fisher’s exact test in DAVID (http:// david.abcc.ncifcrf.gov) (Huang da, Sherman, & Lempicki, 2009; Choi et al., 2012b, 2013a). We previously found that specific biological processes were highly enriched in extracellular vesicles derived from HT29 human colorectal cancer cells (Choi et al., 2012b). Consistent with this observation, the

B cell

Platelet

FIGURE 6. Intravesicular protein–protein interaction networks of mammalian extracellular vesicles. Vesicular proteins are interconnected via physical interactions, and the overall topologies of protein–protein interaction networks of mammalian extracellular vesicles follow the properties of scale-free networks by indicating the presence several hub proteins with many other vesicular proteins having few interacting partners, as reported previously (Choi et al., 2012b, 2013a).

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Enriched biological process in SW480-derived extracellular vesicles

Enriched biological process in B cell-derived extracellular vesicles

Enriched biological process in Platelet-derived extracellular vesicles

Lysosome

Endoplasmic reticulum Golgi apparatus

Nucleus

Cytosol

Platelet

B cell

SW480

Plasma membrane

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Extracellular vesicles

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Protein localization Protein transport Vesicle-mediated transport Protein targeting Protein folding Regulation of protein modification process Regulation of protein ubiquitination Membrane organization Protein complex biogenesis Regulation of organelle organization Cytoskeleton organization Actin cytoskeleton organization Regulation of cell death Cell adhesion Cell motion Cell cycle Response to protein stimulus Small GTPase mediated signal transduction Generation of precursor metabolites and energy Glycolysis Cell-cell junction organization Cell-cell junction assembly Cell morphogenesis Cell morphogenesis involved in differentiation Cell projection morphogenesis Cell adhesion mediated by integrin Leukocyte activation Lymphocyte activation T cell activation Regulation of body fluid levels Hemostasis P-value Wound healing Coagulation Platelet activation 1 10-1 10-2 10-3 10-4

FIGURE 7. Comparison of extracellular vesicles with other subcellular organelles or compartments. The functional annotations of vesicular proteins with specific biological processes are highly enriched or depleted in three mammalian extracellular vesicles when compared with other intracellular organelles or compartments. Colors indicate the statistical significance of enriched or depleted Gene Ontology biological process annotations. The vesicular protein list of enriched or depleted biological processes is available from EVpedia (http://evpedia. info) as Supplementary Table.

functional annotations of vesicular proteins indicate that specific biological processes are highly enriched or depleted in three mammalian extracellular vesicles when compared with other intracellular compartments (Fig. 7). The vesicular protein list of enriched or depleted biological processes is available from EVpedia (http://evpedia.info) as Supplementary Table. All three extracellular vesicles are highly enriched with the following vesicle structure- and vesicle biogenesis-related biological processes: protein localization, protein transport, vesicle-mediated transport, protein targeting, regulation of protein ubiquitination, membrane organization, protein complex biogenesis,

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regulation of organelle organization, cytoskeleton organization, actin cytoskeleton organization, and small GTPase-mediated signal transduction (Raimondo et al., 2011; Choi et al., 2012b, 2013a). These results suggest that extracellular vesicles are extracellular organelles that are distinct from other intracellular organelles and compartments (Choi et al., 2012b, 2013a). Interestingly, SW480-, B cell- and platelet-derived extracellular vesicles are enriched with cell-type-specific biological processes. For example, extracellular vesicles that are derived from SW480 human colorectal cancer cells are specifically enriched with cell-cell junction assembly, cell morphogenesis,

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cell morphogenesis involved in differentiation, cell projection morphogenesis, and cell adhesion mediated by integrin processes. These are well-known biological processes that are involved in epithelial-mesenchymal transitions in human cancer (Yang & Weinberg, 2008; Choi et al., 2012a). B cell-derived extracellular vesicles are enriched with leukocyte, lymphocyte, and T cell activation-associated proteins, whereas platelet-derived extracellular vesicles are specifically enriched with the regulation of body fluid levels, hemostasis, wound healing, coagulation, and platelet activation. These observations suggest that extracellular vesicles contain not only the common sets of vesicular proteins but also cell-type-specific proteins that are associated with celltype-specific pathophysiological functions (de Gassart et al., 2004; Choi et al., 2013a).

C. Functional Modules in the Intravesicular Protein–Protein Interaction Networks of Mammalian Extracellular Vesicles Based on functional enrichment analyses (Fig. 7) and literature mining (Choi et al., 2012b), we constructed subnetworks to

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define common functional modules that are clustered with vesicle-enriched common biological processes: vesicle-mediated transport (Fig. 8A) and cytoskeleton organization (Fig. 8B). The functional modules of the coatomer family, ESCRTs, exocytosis, RAB proteins, and 14-3-3 proteins are clustered in the vesicle-mediated transport subnetwork (Fig. 8A), whereas the cytoskeleton organization-related subnetwork is clustered with functional modules of the actin cytoskeleton, Arp2/3 proteins, and ezrin/radixin/moesin (Fig. 8B). These common functional modules in the vesicle-mediated transport and cytoskeleton organization-related subnetworks are known to be involved in the biogenesis and structure of extracellular vesicles (Laulagnier et al., 2004, 2005; Choi et al., 2012b, 2013a). We also found that three different extracellular vesicles harbor cell-type-specific functional modules that are related by physical and functional interconnections (Fig. 8C). The functional modules of junction proteins, leukocyte activation, and coagulation are only present in extracellular vesicles that are derived from SW480 human colorectal cancer cells, B cells, and platelets, respectively. Junction proteins are important for maintaining the cellular integrity of and polarity in intestinal epithelial cells, and a loss of normal junctions could be a

FIGURE 8. Functional modules clustered with enriched biological processes in mammalian extracellular vesicles. Common functional modules in the vesicle-mediated transport subnetworks (A) and cytoskeleton organization-related subnetworks (B) are present in all extracellular vesicles that are derived from SW480 human colorectal cancer cells, B cells, and platelets. These common functional modules are known to be involved in the biogenesis and structure of extracellular vesicles (Laulagnier et al., 2004, 2005; Choi et al., 2012b, 2013a). C: Cell-type-specific functional modules in mammalian extracellular vesicles. The functional modules of junction proteins, leukocyte activation, and coagulation are only present in extracellular vesicles derived from SW480 human colorectal cancer cells, B cells, and platelets, respectively.

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hallmark of a human carcinoma (Conacci-Sorrell, Zhurinsky, & Ben-Ze’ev, 2002; Martin & Jiang, 2009; Dusek & Attardi, 2011). Moreover, the abnormal regulation of the junction proteins leads to a loss of epithelial polarity and an overproliferation of cells (Lu & Bilder, 2005). It has been reported that B cell-derived extracellular vesicles play roles in immune modulation, such as B cell and T cell activation (McLellan, 2009). For example, MS4A1 and CD22 are involved in B cell activation, whereas CD48 and SLAMF1 are involved in T cell activation. B cell-derived extracellular vesicles play roles in immune modulation, such as B cell and T cell activation (McLellan, 2009). For example, MS4A1 and CD22 are involved in B cell activation, whereas CD48 and SLAMF1 are involved in T cell activation. Platelet-derived extracellular vesicles contain the coagulation-associated functional module. This result is consistent with the previous observation that procoagulant enzymatic activity is one of the main pathophysiological functions of platelet-derived extracellular vesicles (Cocucci, Racchetti, & Meldolesi, 2009). Taken together, all these observations suggest that extracellular vesicles contain common and cell-type-specific functional modules.

V. CONCLUSIONS AND PERSPECTIVES Extracellular vesicle-mediated communication is an evolutionarily conserved process among bacteria, archaea, and eukaryotes (Lee et al., 2008, 2009; Deatherage & Cookson, 2012; Choi et al., 2013a). Growing evidence indicates that these extracellular vesicles, including mammalian exosomes and ectosomes, are extracellular organelles (i.e., nanocosmoses) that play diverse roles in intercellular communication in multicellular communities. In this review, we focused on the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles (i.e., exosomes and ectosomes), introduced an integrated database of high-throughput data for the systematic analyses of extracellular vesicles (EVpedia), and highlighted intravesicular protein–protein interaction network analyses. As purification technologies and proteomic analytical methods have been developed, more reliable vesicular proteomes have been identified. Moreover, a systems biology approach via protein–protein interaction network analysis provides the functional relationship between these vesicular proteins (Choi et al., 2012b, 2013a). These approaches provide comprehensive information for understanding vesicle biogenesis and cargo-sorting mechanisms as well as their pathophysiological roles. Furthermore, extracellular vesicles can be isolated from various biological fluids with enriched surface proteins of their cells of origin, leading to the discovery of biomarkers. Together with conventional biological research, advanced proteomic analyses with the quantitation of vesicular proteins will provide new knowledge and aid in the comprehension of the complex extracellular vesicle-mediated intercellular communication system. However, the identification of vesicular proteins with high confidence will be critical for the global understanding of the emerging biology of extracellular vesicles. Previous highthroughput mass spectrometry-based proteomic studies have identified several hundred or, in some cases, more than 1,000, vesicular proteins. Moreover, the list and number of identified vesicular proteins varies between proteomic studies that were conducted on extracellular vesicles derived from the same cells

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or body fluids (Choi et al., 2012a; Ji et al., 2013). These observations raise an issue regarding whether these proteins are genuine components of extracellular vesicles because there is a concern that falsely detected proteins could be included in the single proteomics dataset (Stuart et al., 2007; Choi et al., 2012b). First, the isolation of mammalian extracellular vesicles without any contamination by non-vesicular components, including serum abundant proteins, apoptotic bodies, and/or protein aggregates, is a critical prerequisite for high-throughput mass spectrometry-based proteomic analyses (Choi et al., 2011). Therefore, after the isolation of extracellular vesicles, the characterization of the purified extracellular vesicles to indicate their presence without other non-vesicular contaminants should be essential to identify the highly confident vesicular proteins by subsequent mass spectrometry-based proteomic analysis (Fig. 5). The combination of different types of vesicle isolation technologies or repetitive density gradient ultracentrifugation steps should be a promising isolation method to purify the extracellular vesicles without any of the potential contaminations that have been previously reported (Choi et al., 2011; Tauro et al., 2013). In addition, to be more confident that the identified proteins are components of extracellular vesicles, researchers will need to acquire more than two proteomic datasets including quantitative proteomic analyses. Recently, we provided a platform for defining candidate vesicular proteins by integrating proteomic data using genomics and systems approaches (Choi et al., 2012b). From the 1,364 proteins that were identified from five proteomic analyses of human colorectal cancer cell-derived extracellular vesicles, we defined 957 vesicular proteins that are expressed in parental cells based on microarray data and are also interconnected proteins that form an intravesicular protein– protein interaction network. We believe that this approach will provide a rationale for cataloging the genuine proteins within extracellular vesicles that are derived from various types of cells and body fluids. The other important issue that is raised by current proteomic studies is the uncertainty of how many different types of extracellular vesicles are secreted by a single cell. Considering the size of mammalian extracellular vesicles, the presence of several vesicular proteins and nucleic acids, including mRNAs and miRNAs, suggests the possibility that a single cell secretes many different types of extracellular vesicles (Choi et al., 2012b). Further studies to either provide experimental evidence of how many proteins an extracellular vesicle can harbor or determine how many vesicular proteins are present in a single type of extracellular vesicles or in a single extracellular vesicle will be of great value to understand the pathophysiological roles of mammalian extracellular vesicles as well as their biogenesis and cargo-sorting mechanisms (Choi et al., 2012b).

VI. ABBREVIATIONS APEX ESCRT MVB

absolute protein expression endosomal sorting complexes required for transport multivesicular body

ACKNOWLEDGMENTS We thank to Yae Jin Yoon, Ji Hyun Kim, Su Chul Jang, KyongSu Park, Oh Youn Kim, Jaewook Lee, Sae Rom Kim, and

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Gyeongyun Go for manual curation of publications on extracellular vesicle studies in Figure 2. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MEST) (no. 2013035248 and no. 2012R1A1A2042534), the Ministry of Health and Welfare grant funded by the Korea Government (no. A120273), and a grant Korea Basic Science Institute (D33400).

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Dong-Sic Choi received B.Sc. and Ph.D. degrees in the Department of Life Sciences from Pohang University of Science and Technology, Republic of Korea, in 2006 and 2012, respectively. He is now a Research Assistant Professor at Pohang University of Science and Technology, Republic of Korea. His current research areas focus on the proteomic analysis and systems biology of extracellular vesicles derived from eukaryotes and prokaryotes and clinical applications based on extracellular vesicles, including biomarker discovery, diagnosis tool development, and drug delivery systems. Dae-Kyum Kim received a B.Sc. degree in the Department of Life Sciences from Pohang University of Science and Technology, Republic of Korea, in 2009. He is now a Ph.D. Candidate at Pohang University of Science and Technology, Republic of Korea. His current research areas focus on the high-throughput analysis and systems biology of extracellular vesicles derived from eukaryotes and prokaryotes. More recently, he reported a study regarding the integrated database of high-throughput data for the systematic analysis of extracellular vesicles: EVpedia (http://evpedia.info). Yoon-Keun Kim, M.D., Ph.D. is a physician scientist. He received his M.D. from Seoul National University in 1987 and received his Ph.D. in 1997. From 2002 to 2003, he was a Visiting Assistant Professor at Yale University, USA. He is currently a Professor in the Department of Life Sciences at the Pohang University of Science and Technology, Republic of Korea. His research interest is the pathogenesis of allergic diseases and chronic inflammatory diseases, such as asthma, atopic dermatitis, diabetes, obesity, gastritis, and inflammatory bowel diseases. His research focuses on the physiological relevance of the pathogenesis of allergic diseases, and he published several papers on the immunopathogenesis of asthma and allergic diseases. More recently, his research has focused on the relationship between extracellular vesicles from bacteria and inflammatory and metabolic diseases. He has reported several studies regarding the medical importance of bacteria-derived extracellular vesicles on the pathogenesis of immune-based inflammatory diseases. Yong Song Gho received his B.Sc. and M.Sc. degrees in Chemistry from Seoul National University, Republic of Korea, in 1987 and 1989, respectively. He obtained his Ph.D. degree in Biochemistry and Biophysics from the University of North Carolina at Chapel Hill, USA, in 1997. Currently, he is an Associate Professor in the Department of Life Sciences at the Pohang University of Science and Technology, Republic of Korea. His group is primarily studying the genomic and proteomic components of extracellular vesicles using systems biology approaches and the pathophysiological functions of the extracellular vesicles derived from eukaryotes and prokaryotes. He is now serving as an Executive Board Member of the “International Society of Extracellular Vesicles (ISEV)” and as the Editor-in-Chief of the “Journal of Extracellular Vesicles” launched by the ISEV.

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