Reviews pubs.acs.org/jpr
Mass-Spectrometry-Based Molecular Characterization of Extracellular Vesicles: Lipidomics and Proteomics Simion Kreimer,†,‡ Arseniy M. Belov,†,‡ Ionita Ghiran,∥ Shashi K. Murthy,†,§ David A. Frank,⊥,# and Alexander R. Ivanov*,†,‡ †
Barnett Institute of Chemical and Biological Analysis, ‡Department of Chemistry and Chemical Biology, and §Department of Chemical Engineering, Northeastern University, Boston, Massachusetts 02115, United States ∥ Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, United States ⊥ Department of Medical Oncology, Dana−Farber Cancer Institute, Boston, Massachusetts 02115, United States # Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, United States ABSTRACT: This review discusses extracellular vesicles (EVs), which are submicron-scale, anuclear, phospholipid bilayer membrane enclosed vesicles that contain lipids, metabolites, proteins, and RNA (micro and messenger). They are shed from many, if not all, cell types and are present in biological fluids and conditioned cell culture media. The term EV, as coined by the International Society of Extracellular Vesicles (ISEV), encompasses exosomes (30−100 nm in diameter), microparticles (100−1000 nm), apoptotic blebs, and other EV subsets. EVs have been implicated in cell−cell communication, coagulation, inflammation, immune response modulation, and disease progression. Multiple studies report that EV secretion from disease-affected cells contributes to disease progression, e.g., tumor niche formation and cancer metastasis. EVs are attractive sources of biomarkers due to their biological relevance and relatively noninvasive accessibility from a range of physiological fluids. This review is focused on the molecular profiling of the protein and lipid constituents of EVs, with emphasis on massspectrometry-based “omic” analytical techniques. The challenges in the purification and molecular characterization of EVs, including contamination of isolates and limitations in sample quantities, are discussed along with possible solutions. Finally, the review discusses the limited but growing investigation of post-translational modifications of EV proteins and potential strategies for future in-depth molecular characterization of EVs. KEYWORDS: extracellular vesicle, EV, exosome, microvesicle, microparticle, molecular profiling, mass spectrometry, proteomics, lipidomics, post-translational modifications
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INTRODUCTION The ability to detect disease early in its progression is necessary to improve the odds for successful treatment, survival, and quality of life. It is increasingly clear that there is an urgent need to develop new noninvasive or minimally invasive diagnostic assays for early disease detection, disease risk assessment, and patient therapy response monitoring. Extracellular vesicles (EVs) have been isolated from diverse body fluids, including blood plasma and serum,1 urine,2 pleural effusions,3 bronchial fluid,4 synovial fluid,5 ocular fluids,6 breast milk,7 human saliva,8 and cerebrospinal fluid.9 Contrary to the mechanisms of molecular transport via intracellular vesicles that have been well-characterized and recognized by the 2013 Nobel Prize in Physiology or Medicine awarded to Rothman, Schekman, and Südhof,10 the functions of EVs and the mechanisms of their transport are not completely understood. The growing number of publications on EVs demonstrates that this field has begun to bring increasingly more attention to the structural and functional biology of EVs. The International Society of Extracellular Vesicles (ISEV), the American Society for © XXXX American Chemical Society
Exosomes and Microvesicles (ASEMV), and the Journal of Extracellular Vesicles have been established to advance the field of EV research. Furthermore, three curated data repositories have been established to help disseminate proteomic, lipidomic, and transcriptomic EV data: ExoCarta,11 Vesiclepedia,12 and EVpedia.13 Much of the interest in EVs stems from their accessibility from physiological fluids and their proven involvement in biological processes such as tumor niche formation,14 metastasis,15 and immune system modulation,16,17 which highlights their potential as minimally invasive biomarker sources. The term EV was coined to encompass extracellular membrane-enclosed vesicles,18 which have been previously described as vaults,19 microsomes,20 lipid vesicles,21 ectosomes,22 microvesicles,23 microparticles,24 exosomes,25 and oncosomes.26 In many publications, distinctions are made between multivesicular-body-derived exosomes with a diameter Received: December 12, 2014
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DOI: 10.1021/pr501279t J. Proteome Res. XXXX, XXX, XXX−XXX
Reviews
Journal of Proteome Research
Table 1. Overlap in Physical Attributes of Particles and Vesicular Structures That May Be Co-isolated in Microparticle or Exosome Preparationsa particle name
description
diameter (nm)
density (g/mL)
exosomes
vesicles released from MVBs
30−120
1.13−1.21
ectosomes, microparticles, and microvesicles apoptotic blebs
vesicles formed by direct budding from cell membrane
50−1000
∼1.16
apoptosis fragments
50−500
1.16−1.28
large oncosomesb
nonapoptotic plasma membrane blebs shed by “amoeboid” migrating tumor cells secreted RNA−protein complexes
1000−10,000
immunocomplexes viral particles
lipoprotein particles (LP)
argonaute complexesc
markers
references
Alix, TSG101, HSC70, CD63, CD81, CD9, among others surface phosphatidylserine, selectins, integrins, CD40, metalloproteases
Mathivan (2010)42 Barteneva (2013), Choi (2007)43,44 Mathivan (2010)42
N/A
surface phosphatidylserine, histones, calnexin, cytochrome C Cav-1, ARF6
∼12
N/A
Ago 2, miRNA
protein clusters containing immunoglobulins influenza HIV-1
50−250 85−120 1113−139
>1.21 N/A 1.16−1.18
immunoglobulins hemagglutinin Gp120, gp41, gp32, host MHC-II
very-low-density LP-1
33−70
100000g).56 Nonpolymerized THP is also a major component of the ultracentrifugation EV pellet. The addition of DTT during the isolation step has been shown to reduce the loss of EV sample in the low-gravity centrifugation pellet by breaking apart the THP networks, but is ineffective in removing the protein from the high-gravity pellet.56 Additionally, the presence of reducing agents in the isolate can lead to protein denaturation and increased aggregation. So-called “insoluble immunocomplexes” are formed by the aggregation of IgG, IgM, and associated proteins during the preparation of plasma-derived EV samples. These particles are prevalent in patients with rheumatoid arthritis and other autoimmune disorders. The immunocomplex size range overlaps with both the microparticle and the exosome ranges. Immunocomplexes introduce high levels of immunoglobulins to the high-gravity EV pellets47 and reduce the detection of EV proteins. Even without aggregation, highabundance proteins infiltrate EV samples in minimal volumes of leftover supernatant and by sticking to container walls or the EVs themselves. Protein contamination can be mitigated by additional sample purification, such as density gradient flotation, or by the immunoaffinity isolation of EV subpopulations.57,58 The separation of intact EVs by hydro-
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LIPIDOMIC CHARACTERIZATION OF EVS Lipidomics involves the comprehensive characterization and quantitation of lipid species in biological samples as well as the assessment of the lipid compositional responses to various stimuli on the cell and organism levels.65 Fundamental biological functions including energy storage, cell membrane structure, temperature maintenance, chemical signaling, and cholesterol metabolism are modulated by lipid species. Lipid species are defined by permutations of the head and tail groups, with various possible modifications that further increase their complexity and heterogeneity; because of this, the complete identification of all lipid species implicated in biological roles in mammalian tissues has not yet been achieved, and new lipid structures are still being discovered.66 However, highthroughput and high-sensitivity mass-spectrometry-based approaches, including liquid chromatography and gas chromatography coupled to MS (LC−MS and GC−MS), are able to provide comprehensive and quantitative assessments of a large repertoire of lipid species in various biological samples, including EVs.37,67 The thickness of a lipid bilayer membrane is roughly 5 nm, which suggests that lipids comprise roughly two-thirds of the volume of the smallest exosomes ∼30 nm in diameter. This ratio rapidly drops for larger EVs, but nonetheless, isolated EV fractions are rich in lipids, which play important biological roles in EVs. Sphingolipid ceramide is implicated in exosomal but not lysosomal MVB formation, lipid-raft domain association is a packaging pathway for EV proteins,68,69 and, recently, the neutral sphingomyelinase pathway was implicated in prion EV packaging.70 To better understand EV biogenesis and protein packaging pathways, and to exploit an additional source of molecular markers, extensive lipidomic profiling studies of EVs are necessary. EV lysis and lipid extraction is typically carried out in a single step through liquid−liquid phase extraction. Analysis of the lipid classes has been accomplished with lysis and extraction using a 4:1 THF:H2O phase followed by the separation of phospholipids and glycophospholipids by diethyl ether and water partitioning.71 Alternatively, EV lipids may be extracted by the Bligh and Dyer liquid−liquid phase extraction, in which most lipid species are dissolved in the organic layer of a chloroform/methanol/water (1:1:1 by volume) solution.72 Lipid species from the organic phase may then be separated and analyzed by GC−MS, LC−MS, or direct infusion electrospray ionization into a high-resolution mass spectrometer. Accurate mass measurement is often insufficient for the unambiguous identification of lipid or metabolite species due to overlap in elemental compositions, and the presence of salt adducts further complicates analysis. Additional information in D
DOI: 10.1021/pr501279t J. Proteome Res. XXXX, XXX, XXX−XXX
Reviews
Journal of Proteome Research
functional conformation. It was also suggested that these lipid−protein complexes are implicated in protein sorting into MVBs, generation of membrane buds, and membrane fission.84 Lipidomic analysis of rat mast-cell-derived and human dendritic-cell-derived exosomes revealed that in comparison to parent cells, exosomes are enriched in sphingomyelin but not in cholesterol, contrary to the lipidomic profile of B-cell-derived exosomes. Fluorescent anisotropy of exosomal membranes revealed that an increase of environmental pH from 5 to 7 resulted in an increase in membrane rigidity.85 This shift in pH occurs during exosome release from MVBs into the extracellular space, and the increase in membrane rigidity explains why exosomes are virtually immune to lipolytic and proteolytic degradation while in circulation. In the same study, NMR analysis confirmed the bilayer organization of exosome membranes. Such membranes possessed lower lateral diffusion and higher trans-bilayer movement of phospholipids than in the plasma membranes of parent cell organelles, such as the endoplasmic reticulum (ER) and Golgi apparatus. Human dendritic cell exosomal membrane analysis found symmetry in lipid dispersion between membrane leaflets, which is contrary to the asymmetrical dispersion in the parent dendritic cell membranes. Lastly, it was suggested in the study that because of the differences in phospholipid symmetry between exosomal membranes of those of follicular dendritic cells, the adsorption of whole exosomes into follicular dendritic cells is favored over the fusion of exosomal and dendritic cell membranes.85 The principle of lipid membrane symmetry may be extended to EVs of other cell types to explain the mechanisms of their adsorption into target cells, but this assumption cannot be made without the measurement of phospholipid symmetry. Lipidomic profiling of EVs could also elucidate other fusion mechanisms and may result in the discovery of differences in EV lipid composition and arrangement that are unique to specific cell types or health and disease states. In a study by Llorente et al. (2013), approximately 280 molecular lipid species were characterized from metastatic prostate cancer cell-line (PC-3)-derived exosomes.86 A quantitative comparison of identified lipids between the EV and parent cell membranes determined that exosomes were significantly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (PS), among others. Overall, an 8.4-fold enrichment of lipid species per mg of protein (65 lipid molecules per protein molecule) was observed in exosomes from PC-3 cells in comparison to cells of origin. Enrichment of glycosphingolipids, and the high degree of sorting of lipid classes and species in the exosome membrane, was hypothesized to contribute to their stability in the extracellular environment.86 Cholesterol, sphingolipids, and PS enrichment in clusters on exosome membranes also contributes to their unique functionality and biogenesis mechanisms. PS clusters on the inner leaflet of the exosomal membrane, for example, are negatively charged and transiently bind to cytosolic protein domains, thereby anchoring these proteins to the membrane. Lipid species composition of exosomal membranes may be dictated by their ability to interact with certain cytosolic proteins and form homogeneous clusters, as is the case with PS and the C-termini of the cytosolic proteins annexin A2 and A4.87 Lipidomic characterization of EVs may elucidate novel biomarkers that may be used for disease diagnostics, prognosis, and treatment. The study by Llorente et al. (2013) identified potential biomarkers in PC-3-derived exosomes.86 These
metabolomic and lipidomic experiments is gained by elution time (compared to known standards), relative isotope intensities, fragmentation (tandem mass spectrometry), and ion mobility measurements.73,74 GC−MS is a well-established platform for lipidomic analysis that enables high temporal resolution for low-molecular-weight lipids (
