proteomics of extracellular vesicles: exosomes and ectosomes

PROTEOMICS OF EXTRACELLULAR VESICLES: EXOSOMES ANDECTOSOMES

Dong-Sic Choi, Dae-KyumKim, Yoon-Keun Kim, and Yong SongGho*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 14 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21420

Almost all bacteria, archaea, and eukaryotic cells shed extracel-lular vesicles either constitutively or in a regulated manner.These nanosized membrane vesicles are spherical, bilayeredproteolipids that harbor specific subsets of proteins, DNAs,RNAs, and lipids. Recent research has facilitated conceptualadvancements in this emerging field that indicate that extracel-lular vesicles act as intercellular communicasomes by transfer-ring signals to their target cell via surface ligands anddelivering receptors and functional molecules. Recent progressin mass spectrometry-based proteomic analyses of mammalianextracellular vesicles derived from diverse cell types and bodyfluids has resulted in the identification of several thousandvesicular proteins that provide us with essential clues to themolecular mechanisms involved in vesicle cargo sorting andbiogenesis. Furthermore, cell-type- or disease-specific vesicularproteins help us to understand the pathophysiological functionsof extracellular vesicles and contribute to the discovery ofdiagnostic and therapeutic target proteins. This review focuseson the high-throughput mass spectrometry-based proteomicanalyses of mammalian extracellular vesicles (i.e., exosomesand ectosomes), EVpedia (a free web-based integrated databaseof high-throughput data for systematic analyses of extracellularvesicles; http://evpedia.info), and the intravesicular protein–protein interaction network analyses of mammalian extracellu-lar vesicles. The goal of this article is to encourage furtherstudies to construct a comprehensive proteome database forextracellular vesicles that will help us to not only decode thebiogenesis and cargo-sorting mechanisms during vesicle forma-tion but also elucidate the pathophysiological roles of thesecomplex extracellular organelles. # 2014 Wiley Periodicals,Inc. Mass Spec Rev 34:474–490, 2015

Keywords: intercellular communicasomes; nanocosmos; intra-vesicular protein–protein interaction networks; proteomics;systems biology

I. INTRODUCTION

Throughout the evolutionary process, bacteria, archaea, andeukaryotic cells have secreted nanosized membrane vesicles intothe extracellular space (Fig. 1) (Lee et al., 2008, 2009; Deatherage&Cookson, 2012; Choi et al., 2013a). These extracellular vesiclesare spherical, bilayered proteolipids that are enriched with variousproteins, nucleic acids, and lipids. Although they have long beenconsidered cellular artifacts or dust, recent progress in this areaindicates that extracellular vesicles are intercellular communica-somes, that is, extracellular organelles that have multifacetedphysiological and pathological functions in intercellular commu-nication as well as inter-species and inter-kingdom communica-tion (Choi et al., 2007, 2011; Lee et al., 2008, 2010; Honget al., 2009). Furthermore, the secretion of extracellular vesiclescould have similar features in different organisms, and extracellu-lar vesicle-mediated intercellular communication is evolutionari-ly conserved (Lee et al., 2009; Deatherage & Cookson, 2012;Choi et al., 2013a). Publications regarding prokaryotic andeukaryotic extracellular vesicles have grown rapidly during thelast 10 years (Fig. 2); this rapid growth indicates that the field ofextracellular vesicles is expanding intensively (Kim et al., 2013).Therefore, the study of extracellular vesicles provides crucialclues for understanding the intercellular communication net-work in living organisms and the evolutionary connectionsamong bacteria, archaea, and eukaryotes.

A. Overview of Mammalian Extracellular Vesicles:Exosomes and Ectosomes

Extracellular vesicles are secreted by various mammalian cellsunder physiological conditions and various disease states,including cancer, in which the release of extracellular vesicles isaberrantly increased (Gyorgy et al., 2011). Two mechanisms ofmammalian extracellular vesicle biogenesis have been sug-gested (Fig. 3A): (1) exosomes that are 50–100 nm in diameterare secreted from the endosomal membrane compartment afterthe fusion of multivesicular bodies (MVBs) with a plasmamembrane or (2) ectosomes (also known as microparticles andmicrovesicles) with a diameter of 100 nm to 1mm that are sheddirectly from the plasma membrane. These two types ofmammalian extracellular vesicles harbor various bioactivematerials including proteins, genetic materials (mRNAs andmiRNAs), and lipids (Fig. 3B). The term exosomes is consistent-ly used to indicate the exocytic vesicles from MVBs, whereasectosomes are known by diverse names, including extracellularmembrane vesicles, membrane particles, exovesicles, nano-particles, microvesicles, microparticles, matrix vesicles, onco-somes, and shedding vesicles (Choi et al., 2013a).

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, 2015, 34, 474–490# 2014 by Wiley Periodicals, Inc.

http://evpedia.info/

Despite apparent differences in the mechanism of biogene-sis between exosomes and ectosomes, it is experimentallydifficult to discriminate exosomes from ectosomes after they aresecreted from cells. Furthermore, there is no clearly descriptivephysical property or molecular marker that can unambiguouslydistinguish exosomes from ectosomes (Deneka et al., 2007;Simons & Raposo, 2009; Choi et al., 2012b). Therefore, we referto these mixed vesicle populations as extracellular vesicles(Fig. 3B) (Choi et al., 2012b).

Upon release, both types of extracellular vesicles circulatein the local extracellular space and travel long distances bydiffusion through body fluids, such as blood and lymph.Extracellular vesicles deliver complex signals via stimulating

target cells (Fig. 3A) using the following mechanisms (Ratajc-zak et al., 2006): (1) inducing receptor-mediated intracellularsignal transduction by surface-expressed or bound ligands, (2)transferring surface receptors to target cells, and (3) deliveringfunctional proteins, RNAs, and lipids into the target cells byfusion with plasma membrane or internalization into theendocytic compartment. These vesicle-mediated intercellularcommunications have several advantages. First, extracellularvesicles display the membrane proteins with a high localconcentration. For example, vesicular ICAM1 has a lowermobility than soluble ICAM1, which likely increases the localdensity and overall avidity of ICAM1 (Lee et al., 2010). Second,extracellular vesicles have an endogenous ability to target their

FIGURE 1. Secretion of extracellular vesicles and extracellular vesicle-mediated intercellular communication isevolutionarily conserved from prokaryotes (archaea, Gram-negative bacteria, and Gram-positive bacteria) toeukaryotes includingmammalian cells.

0

200

400

600

800

1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003 2006 2009 2012

Num

ber o

f pub

licat

ions

Year

Eukaryote Prokaryote

FIGURE 2. Publication trends on prokaryotic and eukaryotic extracellular vesicle studies. Publications onprokaryotic and eukaryotic extracellular vesicles that were manually curated after searching candidate papers inPubMed (http://www.ncbi.nlm.nih.gov/pubmed) using the following keywords exosome�, exovesicle�, “mem-brane 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 membraneblebs” (Kim et al., 2013).

Mass Spectrometry Reviews DOI 10.1002/mas 475

PROTEOMICS OF EXTRACELLULAR VESICLES &

http://www.ncbi.nlm.nih.gov/pubmed

cells of origin. Third, extracellular vesicles can transfer func-tional hydrophobic molecules, such as bioactive lipids andmembrane proteins. For example, vesicular sphingomyelinexhibits angiogenic activity by inducing the migration andproliferation of endothelial cells (Kim et al., 2002). In addition,extracellular vesicles promote the oncogenic transformation oftarget cells by transferring vesicular EGFRvIII (Al-Nedawiet al., 2008). Fourth, luminal components of extracellularvesicles are protected from degradation. For example, RNAs(mRNAs and miRNAs) are present in extracellular vesicles thatare separated from body fluids, which are rich in RNase (Valadiet al., 2007).

B. “Multi-Omics” Research on Extracellular Vesicles

To decode the molecular mechanisms that are involved invesicular cargo sorting and biogenesis as well as the diversephysiological and pathological functions of these complexextracellular organelles, several thousand protein and RNAcomponents of extracellular vesicles that are derived fromvarious cell types and body fluids have been catalogued usingproteomic 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 ofvesicular components have revealed that extracellular vesicles

A

B

: Phosphatidylserine : Sphingomyelin

: Ceramide : Cholesterol

Ligand-receptorinteraction

Fusion

Endocytosis

Nucleus

Ceramide

Sphingomyelin

ESCRTs

SyndecanSyntenin

[Ca2+]

CalpainGelsolin

Actin cytoskeleton

nSMase2

Exosomes

Ectosomes

MVB

TGN

Nucleus

Lipids

Vesicle trafficking-related proteins: Annexins, ARFs, ALIX, ESCRTs, TSG101, RABs

Tetraspanins:CD9, CD63, CD81

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

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

Receptors:EGFRs, FGFRs, PDGFRs

Adhesion proteins:CD44, EPCAM, ICAMs, Integrins

Secreted proteins:Complements, fibronectins, MIF

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

Genetic materialsmRNAsmiRNAs

FIGURE 3. Two types of mammalian extracellular vesicles and their molecular components. A: Based on theirbiogenesis 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 multivesicularbodies with the plasma membrane. Ectosomes (also known as microparticles and microvesicles), which are100 nm to 1mm in diameter, are shed directly from the plasma membrane. Released extracellular vesicles delivercomplex 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 enrichedwith cytoskeletal proteins, cytosolic proteins, secreted proteins, plasma membrane proteins (e.g., adhesionproteins, receptors, tetraspanins, and transporters), and vesicle trafficking-related proteins. Their membranes areenriched with cholesterol, ceramide, phosphatidylserine, and sphingolipids. Moreover, mammalian extracellularvesicles contain vesicle-specific genetic materials, such as DNAs, mRNAs, and miRNAs.

476 Mass Spectrometry Reviews DOI 10.1002/mas

& CHOI ET AL.

harbor a specific subset of proteins, genetic materials (mRNAsand miRNAs), and lipids rather than random cellular compo-nents (Fig. 3B). Moreover, these vesicular proteins, mRNAs,miRNAs, and lipids can be grouped as either common vesicularor cell-type-specific vesicular subsets. The common vesicularsubset of proteins, mRNAs, miRNAs, and lipids provide ussome essential clues regarding the molecular mechanisms thatare involved in vesicle cargo sorting and biogenesis, whereas thecell-type-specific vesicular cargo help us to understand thepathophysiological functions of extracellular vesicles and couldprove 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 massspectrometry-based proteomic analyses of mammalian extracel-lular vesicles (i.e., exosomes and ectosomes). A recent review ofthe proteomic analysis of Gram-negative bacteria extracellularvesicles (also known as outer membrane vesicles) has beenpublished (Lee et al., 2008). In addition, we introduce EVpedia,which is a free web-based integrated database of high-through-put data for the systematic analyses of extracellular vesicles(http://evpedia.info), and we highlight the protein–proteininteraction network analyses of mammalian extracellularvesicles that represent the interrelationship between vesicularproteins and their functional activities. The goal of this review isto encourage further studies on the high-throughput massspectrometry-based proteomics and on the intravesicular pro-tein–protein interaction network analyses of extracellularvesicles that provide us with essential clues to the molecularmechanisms involved in vesicle cargo sorting and biogenesis.Furthermore, cell-type- or disease-specific vesicular proteinshelp us to understand the pathophysiological functions of thesecomplex extracellular organelles and contribute to the discoveryof diagnostic and therapeutic target proteins.

II. BIOGENESIS OF MAMMALIANEXTRACELLULAR VESICLES

A. Exosomes

Exosomes were first observed approximately 30 years ago byelectron microscopy as small intracellular vesicles that containselect plasma membrane proteins in maturing reticulocytes (Panet al., 1985). These small vesicles were segregated within amembrane-bound organelle forming a MVB and were releasedvia fusion of the MVB’s external membrane with the plasmamembrane of the cell (Fig. 3A). This process does not proceedby default or at random, but rather it is highly regulated byspecific modulators. The endosomal sorting complexes requiredfor transport (ESCRTs) have been shown to play a key role inthe generation of vesicles in MVBs via the recognition ofubiquitinylated 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 haveubiquitin-interacting modules that are necessary for the sequen-tial sorting of cargo that is destined for the MVBs. ESCRT-0plays a role in the clustering of ubiquitinylated cargo. ESCRT-1and ESCRT-2 are responsible for membrane budding, andESCRT-3 cleaves the budded membrane by fission. Recently,syndecan heparan sulfate proteoglycans and their cytoplasmicadaptor syntenin have been shown to interact with ALIX, an

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

B. Ectosomes

The first description of ectosomes, also frequently referred to byother terms, such as microparticles or microvesicles, wasreported in 1967 (Wolf, 1967). In human plasma, the fragmentsthat were derived from platelets were identified, and they wereshown to have shapes that resembled small membrane vesicles(Wolf, 1967). It has been suggested that ectosome shedding ispreceded by the budding of protrusions on the plasma membranefollowed 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 increasesdramatically upon stimulation rather than upon release fromresting cells. In particular, an increased level of intracellularCa2þ induces a strong ectosome-shedding response in variouscell types (Pilzer et al., 2005; Ratajczak et al., 2006; Moskovich& Fishelson, 2007). The intracellular Ca2þ concentration altersthe asymmetric phospholipid distribution of the plasma mem-brane by activating scramblase and floppase, resulting in theexposure of phosphatidylserine and phosphatidylethanolaminein the outer leaflet of the plasma membrane (Pap et al., 2009).Moreover, the level of Ca2þ contributes to the degradation of thecytoskeleton via the activation of Ca2þ-dependent proteases,such as calpain and gelsolin, followed by ectosome release (Papet al., 2009). Recently, it was reported that RhoA signaling leadsto the downstream activation of a Rho-associated coiled-coil-containing protein kinase that stimulates the generation ofectosomes to regulate the actin cytoskeleton via cofilin (Li et al.,2012). Moreover, the GTP-binding protein ARF6 modulates therelease of protease-loaded ectosomes (Muralidharan-Chariet al., 2009a). By acting through phospholipase D and ERK,ARF6 activates myosin light chain kinase, and the subsequentphosphorylation of myosin light chain regulates the releaseof ectosomes from invasive cells (Muralidharan-Chari et al.,2009a,b). However, the mechanisms of ectosome biogenesis andthe sorting of proteins into ectosomes are more obscure than themechanisms of exosome biogenesis and the sorting of proteinsinto exosomes.

III. MASS SPECTROMETRY-BASED PROTEOMICANALYSES OF MAMMALIAN EXTRACELLULARVESICLES

Early mass spectrometry-based proteomic analyses of mamma-lian extracellular vesicles mainly focused on the identificationof highly abundant vesicular proteins. In these studies, proteinsfrom purified extracellular vesicles were separated using gelelectrophoresis, and then, abundant proteins were analyzed bymass spectrometry (Thery et al., 1999, 2001; van Niel et al.,2001; Skokos et al., 2003). Using these approaches, some ofthe important vesicular marker proteins, such as tetraspanins,




14-3-3 proteins, actin, annexins, and heat shock proteins, wereidentified, 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 massspectrometry technology have improved, several thousandproteins of extracellular vesicles from various cell typesand body fluids, including serum, urine, saliva, ascites, andbreast milk, have been catalogued by high-throughput massspectrometry-based proteomic studies (Choi et al., 2013a; Kimet al., 2013). The lists of all vesicular proteins that have beenidentified by these studies have been deposited in the free web-based EVpedia (http://evpedia.info) database (Fig. 4) (Choiet al., 2013a; Kim et al., 2013). An overview of the currentmethodological approaches in the high-throughput mass spec-trometry-based proteomic analyses of extracellular vesicles isprovided in Figure 5. Although all of the steps described in thisoverall scheme have influenced the quality of the results, thefollowing two methodologies are critical prerequisites for high-throughput mass spectrometry-based proteomic analyses: (1)efficient isolation of mammalian extracellular vesicles withoutany contamination by non-vesicular components and (2) frac-tionation of peptides or proteins prior to mass spectrometricanalysis to reduce the vesicular proteome complexity.

A variety of vesicle isolation methods, including ultracen-trifugation, immunoaffinity beads, flow field-flow fractionation,and gel filtration, are used for the high-throughput massspectrometry-based proteomic analyses of mammalian extracel-lular vesicles from cell culture medium or body fluids, includingserum, urine, saliva, ascites, and breast milk (Fig. 5). Thedetailed vesicle isolation methods for high-throughput massspectrometry-based proteomic analyses are listed in the EVpe-dia (http://evpedia.info) database (Choi et al., 2013a; Kim

et al., 2013). The most commonly used vesicle isolation methodis ultracentrifugation (Thery et al., 2006; Choi et al., 2013a).After the pre-clearing of cells, cell debris, and large apoptoticbodies by differential centrifugation with or without 0.1–0.2mmfiltration, mammalian extracellular vesicles are pelleted byultracentrifugation or sucrose cushion ultracentrifugation (e.g.,100,000–200,000g) (Hong et al., 2009). The vesicle isolationyield 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 toextracellular vesicles should be co-sedimented by this isolationmethod.

Recent progress in high-throughput mass spectrometry-based proteomic analyses of extracellular vesicles indicates thatbuoyant density gradient ultracentrifugation is one of the bestvesicle isolation methods to remove the above-mentionedcontaminants that are caused by ultracentrifugation- or sucrosecushion ultracentrifugation-based vesicle isolation methods.Recently, several alternative vesicle isolation methods, includ-ing immunoaffinity beads, flow field-flow fractionation, andgel 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, thetechnical standardization for challenging methods that are usedto isolate extracellular vesicles is urgently needed because thelists of vesicular proteins identified by mass spectrometry-basedproteomic analyses are largely dependent on the methods thatare used for the isolation of extracellular vesicles. Recentreviews of this subject are available (Choi et al., 2013a; Witweret al., 2013).

Prior to high-throughput mass spectrometry-based proteo-mic analysis, dynamic range reduction of vesicular proteins or

Database: Vesicular protein, mRNA, miRNA, and lipid

Search: Protein

Browse: Protein, mRNA, miRNA, and lipid

Gene Ontology Analysis Protein and mRNA

Network AnalysisProtein and mRNA

Set Analysis

Analysis

Search and Browse

Prokaryotes Eukaryotes

All Mammals Non-mammalian

eukaryotes

High-throughput proteomesa

Datasets 23 97 9 129

Proteins 3,526 47,759 1,491 52,786

High-throughput transcriptomes

mRNA

Datasets 0 19 0 19

mRNAs 0 164,519 0 164,519

miRNA

Datasets 0 22 0 22

miRNAs 0 13,287 0 13,287

Lipidomes

Datasets 1 17 2 20

Lipids 16 300 29 345

aHigh-throughput proteomes in which at least 50 vesicular proteins were identified (Choi et al., 2013a; Kim et al., 2013).

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


& CHOI ET AL.




peptides by fractionation is critical to identify the highlyconfident vesicular proteins and to catalogue a large number ofvesicular proteins (Choi et al., 2013a). As shown in Figure 5,three main strategies have been used to reduce the complexity ofthe vesicular proteome: (1) sodium dodecyl sulfate–polyacryl-amide gel electrophoresis-based vesicular protein fractionation(Utleg et al., 2003; Pisitkun, Shen, & Knepper, 2004), (2) two-dimensional liquid chromatography-based vesicular peptidefractionation, known as multidimensional protein identificationtechnology (Gonzalez-Begne et al., 2009), and (3) isoelectricfocusing-based fractionation of vesicular proteins or peptides(Choi et al., 2012a).

In the following subsections, we summarize previous massspectrometry-based proteomic studies of mammalian extracel-lular vesicles by introducing an integrated database of high-throughput data for the systematic analyses of extracellularvesicles (EVpedia) and discuss in detail the quantitation-basedcomparative proteomic analyses of mammalian extracellular

vesicles and the roles of proteomics in understanding mammali-an extracellular vesicles.

A. EVpedia: An Integrated Database of High-Throughput Data for the Systematic Analyses ofExtracellular Vesicles

Currently, three public online databases of vesicular proteins,mRNAs, miRNAs, and lipids (EVpedia, ExoCarta, andVesiclepedia) are available. EVpedia provides lists of thecomprehensive vesicular proteins, mRNAs, miRNAs, and lipidsof prokaryotic, non-mammalian eukaryotic, and mammalianextracellular vesicles (Choi et al., 2013a; Kim et al., 2013),while ExoCarta (Mathivanan & Simpson, 2009) and Vesiclepe-dia (Kalra et al., 2012) collects only the vesicular componentsof non-mammalian eukaryotic and mammalian extracellularvesicles. In the current EVpedia database (http://evpedia.info), a

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




total of 230,937 vesicular proteins, mRNAs, miRNAs, and lipidsfrom 190 high-throughput datasets were collected: 47,759vesicular proteins deposited from the 97 high-throughput massspectrometry-based proteomic datasets of mammalian extracel-lular vesicles (Fig. 4). Moreover, EVpedia provides an array ofanalytical tools: (1) search for and browse vesicular compo-nents, (2) Gene Ontology enrichment analysis of vesicularcomponents, (3) network analysis of vesicular components, and(4) set analysis: a comparison of vesicular datasets by orthologidentification. In addition, the detailed methods for the isolationof extracellular vesicles and publications on extracellular vesiclestudies are listed in the database. An overall comparison ofEVpedia with Exocarta and Vesiclepedia has been recentlypublished (Choi et al., 2013a; Kim et al., 2013). By providingthese vesicle-specific components that are derived from varioustypes of cells and body fluids as well as an array of analyticaltools for systematic analyses, these integrated online databasesfacilitate communication and resource sharing between re-searchers in this emerging field of biology. Researchers canupload their high throughput datasets to the EVpedia databasethrough the “Upload” menu (Kim et al., 2013).

B. Quantitation-Based Comparative ProteomicAnalyses of Mammalian Extracellular Vesicles

Although most mammalian cells secrete extracellular vesicles,their release rates and their molecular components, includingproteins, mRNAs, and miRNAs, are dependent on cell type.Furthermore, similar to the cellular proteome, vesicular proteincomponents and their abundance are influenced by cellularstatus, including stimulation by growth factor (Di Vizio et al.,2009), exposure to the aging process (Lehmann et al., 2008), andtransfection with infectious molecules (Coleman et al., 2012).Therefore, quantitation-based comparative proteomic analysesof mammalian extracellular vesicles that are derived fromdiverse cell types and cellular conditions are expected to provideinsights into the emerging biology of extracellular vesicles, toelucidate the biogenesis and pathophysiological functions ofextracellular vesicles and to contribute to the discovery ofdiagnostic and therapeutic target proteins.

Two technical approaches are applied for protein quantita-tion in proteomics: label-based and label-free quantitation(Fig. 5). Label-based quantitation methods, such as isobaric tagsfor relative and absolute quantification (iTRAQ) and stable-isotope labeling of amino acids (SILAC) are commonly used invarious comparative proteomic analyses, but these laboriousapproaches are expensive, multistep labeling processes and arenot always feasible due to the insufficient labeling of tagsaccording to the discrimination of samples (Bachi & Bonaldi,2008). Alternatively, label-free quantitation methods, based onthe extracted chromatograms of spectral intensity or spectralcounts, provide inexpensive, convenient, and reliable methodsfor quantitative proteomics studies (Bachi & Bonaldi, 2008).The spectral counting method is based on the assumption thatmore abundant proteins have more abundant peptides. Theabsolute protein expression (APEX) tool is based on spectralcounting of each protein, and it is corrected by the priorexpectation of observing each peptide (Lu et al., 2007). Thisapproach provides absolute protein concentrations across ap-proximately three to four orders of magnitude. Moreover, theprotein abundance that is quantified by APEX is consistent with

Western blotting and two-dimensional gel electrophoresis (Luet al., 2007).

The currently available high-throughput quantitation-basedcomparative proteomic analyses of mammalian extracellularvesicles are summarized in Table 1. For example, we recentlyconducted APEX-based label-free quantitative proteomic analy-ses of extracellular vesicles derived from human primarycolorectal cancer cells (SW480) and their metastatic derivatives(SW620) (Choi et al., 2012a). From the comparison of theestimated abundance of vesicular proteins, 368 and 359 enrichedproteins were identified in SW480- and SW620-derived extra-cellular vesicles, respectively. These differentially regulatedproteins are closely related to the cellular status. For example,SW620-enriched vesicular proteins are associated with cancerprogression and multidrug resistance as potential diagnosticmarkers of metastatic cancer, whereas SW480-enriched vesicu-lar proteins play a role in cell adhesion.

C. Role of Proteomics in Understanding MammalianExtracellular Vesicles

As more vesicular proteins are identified, it has become apparentthat extracellular vesicles contain a specific subpopulation ofproteins rather than randomly selected molecules from theircells of origin (de Gassart et al., 2004; Choi et al., 2013a).Vesicular proteins are mainly derived from the plasma mem-brane, cytosol, and internal vesicles and not from otherintracellular organelles, such as the endoplasmic reticulum,Golgi apparatus, mitochondria, and nucleus. Moreover, extra-cellular vesicles harbor a common set of vesicular proteins andcell-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 de-hydrogenase, peroxiredoxins, and pyruvate kinase), ribosomalproteins, tetraspanins (CD9, CD63, and CD81), and vesicletrafficking-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 arefound most frequently in mammalian extracellular vesicles. Theidentification count of each vesicular protein represents thenumber of identifications of that protein in high-throughputproteomic datasets (Choi et al., 2013a). EVpedia (http://evpedia.info) provides a long list of vesicular proteins and theiroccurrences within prokaryotic and mammalian extracellularvesicles (Choi et al., 2013a; Kim et al., 2013). In addition,diverse functional proteins, including cell-surface antigens,immune-modulating proteins, proteases, and angiogenic mole-cules, have been identified (Choi et al., 2007). Therefore, thesevesicular proteomes provide diverse information on the natureof extracellular vesicles and help us to decode the molecularmechanisms that are involved in vesicular cargo sorting andbiogenesis as well as the diverse physiological and pathologicalfunctions of these complex extracellular organelles (Choi et al.,2007, 2013a). Finally, high-throughput mass spectrometry-based proteomic studies on extracellular vesicles facilitatebiomarker discovery based on the protein signature of theoriginating cells. Recent reviews of this subject are available inthe literature (Raimondo et al., 2011; Choi et al., 2013a,b).


& CHOI ET AL.



TABLE

1.High-throughputquantitation-based

comparativeproteomicanalysesofmam

malianextracellularvesicles

ESI,electrosprayionization;iTRAQ,isobarictagsforrelativeandabsolutequantification;LC,liquidchromatography;MALDI,matrix-assistedlaserdesorption;MS/M

S,tandem

massspectrometry.

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ratio

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M

ass

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eter

Pr

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n Ye

ar

Ref

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n

(0.1

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µm

) U

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ultra

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radi

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(Wild

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, D

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(Par

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l; he

tero

zygo

us K

RA

S), a

nd

DK

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(Mut

ant K

RA

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+ +

− −

−

+ ES

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l co

unts

20

13

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

noitacifitnedI slobmys eneG eman nietorP count

19 2GTCA ,2LBTCA ,1ATCA ,1CTCA ,2ATCA ,BTCA snietorp detaler dna nitcA 1 ,JETOP ,EETOP ,PKETOP ,1GTCA ,IETOP ,FETOP niryknA 2

POTEB 91

APSH ,A1APSH 07 nietorp kcohs taeH 3 5, HSPA8, HSPA6, HSPA2, HSPA1L, HSPA7

90

28 NSM ,XDR ,RZE niseoM/nixidaR/nirzE 45 Glyceraldehyde-3-phosphate dehydrogenase/erythrose-

4-phosphate dehydrogenase 87 SHDPAG ,HDPAG

67 2P2AXNA ,2AXNA 2A nixennA 6 67 A6LAHDL ,BHDL ,AHDL esanegordyhed etatcal-L 7 67 3XDRP ,2XDRP ,1XDRP nixoderixoreP 8 57 3ONE ,2ONE ,1ONE esalonE 9 27 EAHWY nolispe nietorp 3-3-41 01

,1BBUT ,BBUT ateb nilubuT 11 TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBB4B, TUBB6, TUBB8

72

17 1LFC 1-nilifoC 211AA09PSH 09 nietorp kcohs taeH 31 , HSP90AB1, HSP90B1, HSP90AB3P,

HSP90AB4P, TRAP1 70

14 Translation elongation factor EF-1alpha (G 07 L1SBH ,5P1A1FEE ,2A1FEE ,1A1FEE )esaPT 96 RLKP ,2MKP ,MKP esanik etavuryP 51

ABUT ,A4ABUT ahpla nilubuT 61 1C, TUBA8, TUBA3C, TUBA1A, TUBA1B, TUBA3E, TUBAL3

68

76 2KGP ,1KGP esanik etarecylgohpsohp-3 7118 Guanine nucleotide-binding protein G( 76 3IANG ,2IANG ,1IANG ahpla tinubus )i

56 4NTCA ,3NTCA ,2NTCA ,1NTCA ninitca-ahplA 9120 Ras-related protein 56 C5BAR ,B5BAR 5-baR

46 ZAHWY atled/atez nietorp 3-3-41 12 46 5AXNA 5A nixennA 22 46 CTLC ,1LCTLC niahc yvaeh nirhtalC 32 36 BAHWY ahpla/ateb nietorp 3-3-41 42

1PTA ,1A1PTA esaPTA tropsnart noitaC 52 A2, ATP1A3, ATP1A4, ATP12A, ATP4A 63 36 4BNG ,2BNG ,1BNG nietorp gnidnib-editoelcun eninauG 62 36 1NFP 1-niliforP 72 16 AIPP esaremosi snart-sic lylorp-lyditpeP 82 06 A1BAR A1-baR nietorp detaler-saR 92

30 Programmed cell death 6-inte 95 PI6DCDP nietorp gnitcar 85 QAHWY ateht nietorp 3-3-41 13 75 GAHWY ammag nietorp 3-3-41 23 75 BLA nimubla mureS 33 65 4DHE ,3DHE ,2DHE ,1DHE nietorp gniniatnoc-niamod HE 43 65 B1PAR b1-paR nietorp detaler-saR 53 55 1FRA 1 rotcaf noitalysobir-PDA 63 55 7BGTI ,6BGTI ,5BGTI ,3BGTI ,2BGTI ,1BGTI ateb nirgetnI 73 55 6XDRP 6-nixoderixoreP 83 45 IPG esaremosi etahpsohp-6-esoculG 93 45 PBCDS 1-ninetnyS 04 45 1IPT esaremosi etahpsohpesoirT 14

42 Ubiquitin-like modifier-activa 45 7ABU ,6ABU ,1ABU emyzne gnit 35 6AXNA 6A nixennA 34

44 Cell division control prot 35 24CDC golomoh 24 nie 25 AODLA ,CODLA esalodla etahpsohpsib-6,1-esotcurF 54 25 2DUTFE ,2FEE )sesaPTG( srotcaf noitagnole noitalsnarT 64 15 9DC negitna 9DC 74 15 B11BAR ,A11BAR 11-baR nietorp detaler-saR 84 05 11AXNA ,7AXNA 11/7A nixennA 94


& CHOI ET AL.

TABLE 2. (Continued)

05 1CILC 1 nietorp lennahc ralullecartni edirolhC 05 05 NSAF esahtnys dica yttaF 15

,9HYM ,8HYM ,7HYM ,4HYM ,3HYM ,1HYM niahc yvaeh nisoyM 25MYH10, MYH11, MYH14, MYH13, MYH15

50

05 A7BAR a7-baR nietorp detaler-saR 3554 4F2 cell-surface antigen 94 2A3CLS niahc yvaeh55 Eukaryotic initiation fact 94 2A4FIE ,1A4FIE A4 ro56 Ras-related C3 botulinum t 94 2CAR ,1CAR etartsbus nixo

94 2TCC ateb tinubus 1 nietorp xelpmoc-T 75 84 YCHA esanietsycomohlysonedA 85 84 B5PTA lairdnohcotim ,ateb tinubus esahtnys PTA 95 84 5C ,B4C ,A4C ,3C tnemelpmoC 06

,A1OYM 1 niahc yvaeh nisoyM 16 MYO1B, MYO1C, MYO1D, MYO1E, MYO1F, MYO1G

48

84 2IDG ,1IDG rotibihni noitaicossid PDG baR 26 74 01TRK 01 lateleksotyc I epyt ,nitareK 36

64 Large extracellular alpha-helical 74 PZP ,901DC ,1LM2A ,M2A nietorp 65 Nucleoside diphosphate ki 74 3EMN ,2EMN ,1EMN esan66 Phosphatidylethanolamine-b 74 1PBEP 1 nietorp gnidni

74 01BAR ,B1BAR 01/B1 baR nietorp detaler-saR 76 64 1PTSG P esarefsnart-S enoihtatulG 86 64 1HDM esanegordyhed etalaM 96 64 ALAR A-laR nietorp detaler-saR 07 64 2NLGAT 2-nilegsnarT 17 64 PCV esaPTA muluciter cimsalpodne lanoitisnarT 27 54 YLCA esahtnys etartic-PTA 37

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

DST, FLNA, FLNB, FLNC, LCP1, PLEC, PLS1, PLS3 45

54 1FCAM ,PSD nietorp gnidnib noi muiclaC 57 54 1BPSH 1-ateb nietorp kcohs taeH 67 54 B2PAR ,41BAR 41/B2-baR nietorp detaler-saR 77

,RGF ,KLB esanik nietor enisoryT 87 FYN, HCK, LCK, LYN, SRC, YES1 45 79 6-phosphogluconate dehydroge 44 DGP gnitalyxobraced ,esan

44 18DC negitna 18DC 08 44 ULC niretsulC 18 44 1LIV ,NICS ,NSG nilosleG 28 44 2MAGP ,1MAGP esatum etarecylgohpsohP 38 44 1PCT ahpla tinubus 1 nietorp xelpmoc-T 48

85 Guanine nucleotide-binding pr 34 QANG ,11ANG )q(G nieto86 Guanine nucleotide-binding pr 34 31ANG 31-ahpla tinubus nieto

34 1TRK 1 lateleksotyc II epyt ,nitareK 78 34 3AIDP 3A esaremosi-ediflusid nietorP 88 34 A2BAR A2-baR nietorp detaler-saR 98 34 2NLT ,1NLT nilaT 09 24 MOTS nietorp enarbmem largetni 7 dnab etycorhtyrE 19 24 SANG ,SANG ,LANG sahpla G tinubus nietorp G 29

,A-ALH negitna ytilibitapmocotsiH 39 HLA-B, HLA-C, HLA-E, HLA-H 42 24 A8BAR A8-baR nietorp detaler-saR 49 24 A1V6PTA A tinubus citylatac esaPTA notorp epyt-V 59 14 8AMSP ,7AMSP stinubus ateb dna ahpla,emosaetorp S02 69

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

SPTB, SPTBN1, SPTBN2, SPTBN4, SPTBN5 41

98 Ca2+-binding protein (EF- 14 1NATPS )ylimafrepus dnaH 14 PB3SLAGL nietorp gnidnib-3-nitcelaG 99 14 VAGTI ,5AGTI ,B2AGTI 5/b2-ahpla nirgetnI 001

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




IV. INTRAVESICULAR PROTEIN–PROTEININTERACTION NETWORKS OF MAMMALIANEXTRACELLULAR VESICLES

Although mass spectrometry-based proteomic analyses ofmammalian extracellular vesicles have allowed several thousandvesicular proteins to be cataloged, their physical and functionalinterrelationships remain unclear. Our recent protein–proteininteraction network analyses of mammalian extracellularvesicles indicate that vesicular proteins are physically andfunctionally interconnected to form functional modules in-volved in vesicle biogenesis and pathophysiological functions(Choi et al., 2012b, 2013a). Similar to protein–protein interac-tion networks of other intracellular organelles, includingphagosomes, autophagosomes, and mitochondria, the overalltopology of vesicular protein–protein interaction networksfollows the power-law distribution of the existence of severalhub proteins with other vesicular proteins having few interactingpartners. This observation suggests that extracellular vesiclesare nanocosmos (i.e., a nanosized orderly system of extracellularorganelle), rather than cellular dust (Choi et al., 2012b; Kimet al., 2013).

In this review, we expand our previous observations inextracellular vesicles that are derived from human colorectalcancer cells (SW480), human B cells, and human platelets, andwe discuss the biological meaning of the protein–proteininteraction between their vesicular proteins.

A. Intravesicular Protein–Protein InteractionNetworks of Mammalian Extracellular Vesicles

Using the previously reported vesicular proteomic data ofSW480 (Choi et al., 2012a), B cell (Buschow et al., 2010), andplatelet (Garcia et al., 2005), we first constructed intravesicularprotein–protein interaction networks of mammalian extracellu-lar vesicles (Fig. 6) (Choi et al., 2012b). Protein interaction datawere gathered from BioGRID (version 3.2.101; http://www.thebiogrid.org/), which contains 130,292 physical interactions

between 17,373 proteins. The protein–protein interaction net-works 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 networksby indicating several hub proteins with many other vesicularproteins having few interacting partners. These results suggestthat physically interacting cellular proteins may be co-sortedinto extracellular vesicles, as experimentally validated in ourprevious study (Choi et al., 2012b). Although the detailedmolecular mechanisms by which proteins are loaded intoextracellular vesicles during their biogenesis are not fullyunderstood, increasing evidence indicates that the clustering,oligomerization, or protein–protein interaction among vesicularproteins play critical roles in vesicular cargo sorting (Vidal,Mangeat, & Hoekstra, 1997; Le Naour et al., 2006; Choiet al., 2012b). We previously reported that extracellular vesiclesharbor functional modules that are comprised of physicallyinterconnected tetraspanin web proteins (Choi et al., 2012b).The tetraspanin web is a well-known highly clustered proteincomplex in the cell (Le Naour et al., 2006). Moreover, theantibody-mediated cross-linking of plasma membrane proteins,such as the transferrin receptor in reticulocytes or the MHC II inlymphocytes, induces the sorting of these proteins into extracel-lular vesicles (Vidal, Mangeat, & Hoekstra, 1997).

B. Comparison of Extracellular Vesicles With OtherSubcellular Compartments

As shown in Figure 7, we next conducted analysis to identifybiological processes enriched or depleted in extracellularvesicles relative to other intracellular organelles or compart-ments 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 thatspecific biological processes were highly enriched in extracellu-lar vesicles derived from HT29 human colorectal cancer cells(Choi et al., 2012b). Consistent with this observation, the

B cell Platelet SW480

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


& CHOI ET AL.





functional annotations of vesicular proteins indicate that specificbiological processes are highly enriched or depleted in threemammalian extracellular vesicles when compared with otherintracellular compartments (Fig. 7). The vesicular protein list ofenriched or depleted biological processes is available fromEVpedia (http://evpedia.info) as Supplementary Table. All threeextracellular vesicles are highly enriched with the followingvesicle structure- and vesicle biogenesis-related biologicalprocesses: protein localization, protein transport, vesicle-medi-ated transport, protein targeting, regulation of protein ubiquiti-nation, membrane organization, protein complex biogenesis,

regulation of organelle organization, cytoskeleton organization,actin cytoskeleton organization, and small GTPase-mediatedsignal transduction (Raimondo et al., 2011; Choi et al., 2012b,2013a). These results suggest that extracellular vesicles areextracellular organelles that are distinct from other intracellularorganelles and compartments (Choi et al., 2012b, 2013a).

Interestingly, SW480-, B cell- and platelet-derived extra-cellular vesicles are enriched with cell-type-specific biologicalprocesses. For example, extracellular vesicles that are derivedfrom SW480 human colorectal cancer cells are specificallyenriched with cell-cell junction assembly, cell morphogenesis,

SW

480

B c

ell

Pla

tele

t P

lasm

a m

embr

ane

Cyt

osol

N

ucle

us

End

opla

smic

retic

ulum

G

olgi

app

arat

us

Lyso

som

e Ext

race

llula

r ve

sicl

es

Enriched biological process in B cell-derived

extracellular vesicles

Enriched biological process in Platelet-derived


Enriched biological process in SW480-derived


P-value

1 10-1 10-4 10-2 10-3

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 Wound healing Coagulation Platelet activation

FIGURE 7. Comparison of extracellular vesicles with other subcellular organelles or compartments. Thefunctional annotations of vesicular proteins with specific biological processes are highly enriched or depleted inthree 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.






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

C. Functional Modules in the IntravesicularProtein–Protein Interaction Networks ofMammalian Extracellular Vesicles

Based on functional enrichment analyses (Fig. 7) and literaturemining (Choi et al., 2012b), we constructed subnetworks to

define common functional modules that are clustered withvesicle-enriched common biological processes: vesicle-mediat-ed 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 inthe vesicle-mediated transport subnetwork (Fig. 8A), whereasthe cytoskeleton organization-related subnetwork is clusteredwith functional modules of the actin cytoskeleton, Arp2/3proteins, and ezrin/radixin/moesin (Fig. 8B). These commonfunctional modules in the vesicle-mediated transport andcytoskeleton organization-related subnetworks are known to beinvolved 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 vesiclesharbor cell-type-specific functional modules that are related byphysical and functional interconnections (Fig. 8C). The func-tional modules of junction proteins, leukocyte activation, andcoagulation are only present in extracellular vesicles that arederived from SW480 human colorectal cancer cells, B cells, andplatelets, respectively. Junction proteins are important formaintaining the cellular integrity of and polarity in intestinalepithelial cells, and a loss of normal junctions could be a

FIGURE 8. Functional modules clustered with enriched biological processes in mammalian extracellularvesicles. Common functional modules in the vesicle-mediated transport subnetworks (A) and cytoskeletonorganization-related subnetworks (B) are present in all extracellular vesicles that are derived from SW480 humancolorectal cancer cells, B cells, and platelets. These common functional modules are known to be involved in thebiogenesis 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 junctionproteins, leukocyte activation, and coagulation are only present in extracellular vesicles derived from SW480human colorectal cancer cells, B cells, and platelets, respectively.


& CHOI ET AL.

hallmark of a human carcinoma (Conacci-Sorrell, Zhurinsky, &Ben-Ze’ev, 2002; Martin & Jiang, 2009; Dusek & Attardi,2011). Moreover, the abnormal regulation of the junctionproteins leads to a loss of epithelial polarity and an over-proliferation of cells (Lu & Bilder, 2005). It has been reportedthat B cell-derived extracellular vesicles play roles in immunemodulation, such as B cell and T cell activation (McLellan,2009). For example, MS4A1 and CD22 are involved in B cellactivation, whereas CD48 and SLAMF1 are involved in T cellactivation. B cell-derived extracellular vesicles play roles inimmune modulation, such as B cell and T cell activation(McLellan, 2009). For example, MS4A1 and CD22 are involvedin B cell activation, whereas CD48 and SLAMF1 are involved inT cell activation. Platelet-derived extracellular vesicles containthe coagulation-associated functional module. This result isconsistent with the previous observation that procoagulantenzymatic activity is one of the main pathophysiologicalfunctions of platelet-derived extracellular vesicles (Cocucci,Racchetti, & Meldolesi, 2009). Taken together, all theseobservations suggest that extracellular vesicles contain commonand cell-type-specific functional modules.

V. CONCLUSIONS AND PERSPECTIVES

Extracellular vesicle-mediated communication is an evolution-arily conserved process among bacteria, archaea, and eukaryotes(Lee et al., 2008, 2009; Deatherage & Cookson, 2012; Choiet al., 2013a). Growing evidence indicates that these extracellu-lar vesicles, including mammalian exosomes and ectosomes, areextracellular organelles (i.e., nanocosmoses) that play diverseroles in intercellular communication in multicellular communi-ties. In this review, we focused on the high-throughput massspectrometry-based proteomic analyses of mammalian extracel-lular vesicles (i.e., exosomes and ectosomes), introduced anintegrated database of high-throughput data for the systematicanalyses of extracellular vesicles (EVpedia), and highlightedintravesicular protein–protein interaction network analyses.

As purification technologies and proteomic analyticalmethods have been developed, more reliable vesicular pro-teomes have been identified. Moreover, a systems biologyapproach via protein–protein interaction network analysisprovides the functional relationship between these vesicularproteins (Choi et al., 2012b, 2013a). These approaches providecomprehensive information for understanding vesicle biogene-sis and cargo-sorting mechanisms as well as their pathophysio-logical roles. Furthermore, extracellular vesicles can be isolatedfrom various biological fluids with enriched surface proteins oftheir cells of origin, leading to the discovery of biomarkers.Together with conventional biological research, advancedproteomic analyses with the quantitation of vesicular proteinswill provide new knowledge and aid in the comprehension of thecomplex extracellular vesicle-mediated intercellular communi-cation system.

However, the identification of vesicular proteins with highconfidence will be critical for the global understanding of theemerging biology of extracellular vesicles. Previous high-throughput mass spectrometry-based proteomic studies haveidentified several hundred or, in some cases, more than 1,000,vesicular proteins. Moreover, the list and number of identifiedvesicular proteins varies between proteomic studies that wereconducted on extracellular vesicles derived from the same cells

or body fluids (Choi et al., 2012a; Ji et al., 2013). Theseobservations raise an issue regarding whether these proteins aregenuine components of extracellular vesicles because there is aconcern that falsely detected proteins could be included in thesingle proteomics dataset (Stuart et al., 2007; Choi et al.,2012b). First, the isolation of mammalian extracellular vesicleswithout any contamination by non-vesicular components, in-cluding serum abundant proteins, apoptotic bodies, and/orprotein aggregates, is a critical prerequisite for high-throughputmass spectrometry-based proteomic analyses (Choi et al.,2011). Therefore, after the isolation of extracellular vesicles, thecharacterization of the purified extracellular vesicles to indicatetheir presence without other non-vesicular contaminants shouldbe essential to identify the highly confident vesicular proteinsby subsequent mass spectrometry-based proteomic analysis(Fig. 5). The combination of different types of vesicle isolationtechnologies or repetitive density gradient ultracentrifugationsteps should be a promising isolation method to purify theextracellular vesicles without any of the potential contamina-tions that have been previously reported (Choi et al., 2011; Tauroet al., 2013).

In addition, to be more confident that the identified proteinsare components of extracellular vesicles, researchers will needto acquire more than two proteomic datasets including quantita-tive proteomic analyses. Recently, we provided a platform fordefining candidate vesicular proteins by integrating proteomicdata using genomics and systems approaches (Choi et al.,2012b). From the 1,364 proteins that were identified from fiveproteomic analyses of human colorectal cancer cell-derivedextracellular vesicles, we defined 957 vesicular proteins that areexpressed in parental cells based on microarray data and are alsointerconnected proteins that form an intravesicular protein–protein interaction network. We believe that this approach willprovide a rationale for cataloging the genuine proteins withinextracellular vesicles that are derived from various types of cellsand body fluids.

The other important issue that is raised by current proteo-mic studies is the uncertainty of how many different types ofextracellular vesicles are secreted by a single cell. Consideringthe size of mammalian extracellular vesicles, the presence ofseveral vesicular proteins and nucleic acids, including mRNAsand miRNAs, suggests the possibility that a single cell secretesmany different types of extracellular vesicles (Choi et al.,2012b). Further studies to either provide experimental evidenceof how many proteins an extracellular vesicle can harbor ordetermine how many vesicular proteins are present in a singletype of extracellular vesicles or in a single extracellular vesiclewill be of great value to understand the pathophysiological rolesof mammalian extracellular vesicles as well as their biogenesisand cargo-sorting mechanisms (Choi et al., 2012b).

VI. ABBREVIATIONS

APEX absolute protein expressionESCRT endosomal sorting complexes required for transportMVB multivesicular body

ACKNOWLEDGMENTS

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



Gyeongyun Go for manual curation of publications on extracel-lular vesicle studies in Figure 2. This work was supported by theNational Research Foundation of Korea (NRF) grant fundedby the Korea Government (MEST) (no. 2013035248 and no.2012R1A1A2042534), the Ministry of Health andWelfare grantfunded by the Korea Government (no. A120273), and a grantKorea Basic Science Institute (D33400).

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Dong-Sic Choi received B.Sc. and Ph.D. degrees in the Department of Life Sciences fromPohang 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 andTechnology, Republic of Korea. His current research areas focus on the proteomic analysis andsystems biology of extracellular vesicles derived from eukaryotes and prokaryotes and clinicalapplications based on extracellular vesicles, including biomarker discovery, diagnosis tooldevelopment, and drug delivery systems.

Dae-Kyum Kim received a B.Sc. degree in the Department of Life Sciences from PohangUniversity of Science and Technology, Republic of Korea, in 2009. He is now a Ph.D. Candidateat Pohang University of Science and Technology, Republic of Korea. His current research areasfocus on the high-throughput analysis and systems biology of extracellular vesicles derivedfrom eukaryotes and prokaryotes. More recently, he reported a study regarding the integrateddatabase 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 SeoulNational University in 1987 and received his Ph.D. in 1997. From 2002 to 2003, he was aVisiting Assistant Professor at Yale University, USA. He is currently a Professor in theDepartment of Life Sciences at the Pohang University of Science and Technology, Republic ofKorea. His research interest is the pathogenesis of allergic diseases and chronic inflammatorydiseases, such as asthma, atopic dermatitis, diabetes, obesity, gastritis, and inflammatory boweldiseases. His research focuses on the physiological relevance of the pathogenesis of allergicdiseases, and he published several papers on the immunopathogenesis of asthma and allergicdiseases. More recently, his research has focused on the relationship between extracellularvesicles from bacteria and inflammatory and metabolic diseases. He has reported several studiesregarding the medical importance of bacteria-derived extracellular vesicles on the pathogenesisof immune-based inflammatory diseases.

Yong Song Gho received his B.Sc. and M.Sc. degrees in Chemistry from Seoul NationalUniversity, Republic of Korea, in 1987 and 1989, respectively. He obtained his Ph.D. degree inBiochemistry and Biophysics from the University of North Carolina at Chapel Hill, USA, in1997. Currently, he is an Associate Professor in the Department of Life Sciences at the PohangUniversity of Science and Technology, Republic of Korea. His group is primarily studying thegenomic and proteomic components of extracellular vesicles using systems biology approachesand the pathophysiological functions of the extracellular vesicles derived from eukaryotes andprokaryotes. He is now serving as an Executive Board Member of the “International Society ofExtracellular Vesicles (ISEV)” and as the Editor-in-Chief of the “Journal of ExtracellularVesicles” launched by the ISEV.


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proteomics of extracellular vesicles: exosomes and ectosomes

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