JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
EVpedia: A Community Web Portal for Extracellular Vesicles Research.
Dae-Kyum Kim, Jaewook Lee, Sae Rom Kim, Dong-Sic Choi, Yae Jin Yoon, Ji Hyun Kim, Gyeongyun Go, Dinh Nhung, Kahye Hong, Su Chul Jang, Si-Hyun Kim, Kyong-Su Park, Oh Youn Kim, Hyun Taek Park, Ji Hye Seo, Elena Aikawa, Monika Baj-Krzyworzeka, Bas W M van Balkom, Mattias Belting, Lionel Blanc, Vincent Bond, Antonella Bongiovanni, Francesc E Borràs, Luc Buée, Edit I Buzás, Lesley Cheng, Aled Clayton, Emanuele Cocucci, Charles S Dela Cruz, Dominic M Desiderio, Dolores Di Vizio, Karin Ekström, Juan M Falcon-Perez, Chris Gardiner, Bernd Giebel, David W Greening, Julia Christina Gross, Dwijendra Gupta, An Hendrix, Andrew F Hill, Michelle M Hill, Esther Nolte-'t Hoen, Do Won Hwang, Jameel Inal, Medicharla V Jagannadham, Muthuvel Jayachandran, Young-Koo Jee, Malene Jørgensen, Kwang Pyo Kim, Yoon-Keun Kim, Thomas Kislinger, Cecilia Lässer, Dong Soo Lee, Hakmo Lee, Johannes van Leeuwen, Thomas Lener, Ming-Lin Liu, Jan Lötvall, Antonio Marcilla, Suresh Mathivanan, Andreas Möller, Jess Morhayim, François Mullier, Irina Nazarenko, Rienk Nieuwland, Diana N Nunes, Ken Pang, Jaesung Park, Tushar Patel, Gabriella Pocsfalvi, Hernando Del Portillo, Ulrich Putz, Marcel I Ramirez, Marcio L Rodrigues, Tae-Young Roh, Felix Royo, Susmita Sahoo, Raymond Schiffelers, Shivani Sharma, Pia Siljander, Richard J Simpson, Carolina Soekmadji, Philip Stahl, Allan Stensballe, Ewa Stępień, Hidetoshi Tahara, Arne Trummer, Hadi Valadi, Laura J Vella, Sun Nyunt Wai, Kenneth Witwer, María Yáñez-Mó, Hyewon Youn, Reinhard Zeidler, Yong Song Gho.
Bioinformatics
PUBLISHED: 11-13-2014
Show Abstract
Hide Abstract
Extracellular vesicles are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for extracellular vesicle-related publications and vesicular components are currently challenging.
Related JoVE Video
Proteogenomic analysis of the Venturia pirina (Pear Scab Fungus) secretome reveals potential effectors.
J. Proteome Res.
PUBLISHED: 07-09-2014
Show Abstract
Hide Abstract
A proteogenomic analysis is presented for Venturia pirina, a fungus that causes scab disease on European pear (Pyrus communis). V. pirina is host-specific, and the infection is thought to be mediated by secreted effector proteins. Currently, only 36 V. pirina proteins are catalogued in GenBank, and the genome sequence is not publicly available. To identify putative effectors, V. pirina was grown in vitro on and in cellophane sheets mimicking its growth in infected leaves. Secreted extracts were analyzed by tandem mass spectrometry, and the data (ProteomeXchange identifier PXD000710) was queried against a protein database generated by combining in silico predicted transcripts with six frame translations of a whole genome sequence of V. pirina (GenBank Accession JEMP00000000 ). We identified 1088 distinct V. pirina protein groups (FDR 1%) including 1085 detected for the first time. Thirty novel (not in silico predicted) proteins were found, of which 14 were identified as potential effectors based on characteristic features of fungal effector protein sequences. We also used evidence from semitryptic peptides at the protein N-terminus to corroborate in silico signal peptide predictions for 22 proteins, including several potential effectors. The analysis highlights the utility of proteogenomics in the study of secreted effectors.
Related JoVE Video
Extracellular vesicles including exosomes are mediators of signal transduction: Are they protective or pathogenic?
Proteomics
PUBLISHED: 05-24-2014
Show Abstract
Hide Abstract
Extracellular vesicles are signaling organelles that are released by many cell types and is highly conserved in both prokaryotes and eukaryotes. Based on the mechanism of biogenesis, these membranous vesicles can be classified as exosomes, shedding microvesicles and apoptotic blebs. It is becoming clearer that these extracellular vesicles mediate signal transduction in both autocrine and paracrine fashion by the transfer of proteins and RNA. Whilst the role of extracellular vesicles including exosomes in pathogenesis is well established, very little is known about their function in normal physiological conditions. Recent evidences allude that extracellular vesicles can mediate both protective and pathogenic effects depending on the precise state. In this review, we discuss the involvement of extracellular vesicle as mediators of signal transduction in neurodegenerative diseases and cancer. In addition, the role of extracellular vesicles in mediating Wnt and PI3K signaling pathways is also discussed. Additional findings on the involvement of extracellular vesicles in homeostasis and disease progression will promote a better biological understanding, advance future therapeutic and diagnostic applications. This article is protected by copyright. All rights reserved.
Related JoVE Video
Extracellular Vesicles from Neural Stem Cells Transfer IFN-? via Ifngr1 to Activate Stat1 Signaling in Target Cells.
Mol. Cell
PUBLISHED: 02-25-2014
Show Abstract
Hide Abstract
The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-?) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-? bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-?/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-?/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.
Related JoVE Video
Plasma Proteome Database as a resource for proteomics research: 2014 update.
Nucleic Acids Res.
PUBLISHED: 12-03-2013
Show Abstract
Hide Abstract
Plasma Proteome Database (PPD; http://www.plasmaproteomedatabase.org/) was initially described in the year 2005 as a part of Human Proteome Organizations (HUPOs) pilot initiative on Human Plasma Proteome Project. Since then, improvements in proteomic technologies and increased throughput have led to identification of a large number of novel plasma proteins. To keep up with this increase in data, we have significantly enriched the proteomic information in PPD. This database currently contains information on 10 546 proteins detected in serum/plasma of which 3784 have been reported in two or more studies. The latest version of the database also incorporates mass spectrometry-derived data including experimentally verified proteotypic peptides used for multiple reaction monitoring assays. Other novel features include published plasma/serum concentrations for 1278 proteins along with a separate category of plasma-derived extracellular vesicle proteins. As plasma proteins have become a major thrust in the field of biomarkers, we have enabled a batch-based query designated Plasma Proteome Explorer, which will permit the users in screening a list of proteins or peptides against known plasma proteins to assess novelty of their data set. We believe that PPD will facilitate both clinical and basic research by serving as a comprehensive reference of plasma proteins in humans and accelerate biomarker discovery and translation efforts.
Related JoVE Video
Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.
Proteomics
PUBLISHED: 07-10-2013
Show Abstract
Hide Abstract
Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.
Related JoVE Video
Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components.
Proteomics
PUBLISHED: 03-08-2013
Show Abstract
Hide Abstract
Exosomes are small extracellular 40-100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist premetastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signaling molecules fundamental to tumor progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40-100 nm in diameter, were of a buoyant density ~1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix, and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, JAG1, SRC, TNIK), and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalization of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in colorectal cancer cell exosomes. The selective enrichment of metastatic factors and signaling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the cross-talk between tumor and stromal cells in the tumor microenvironment.
Related JoVE Video
ExoCarta 2012: database of exosomal proteins, RNA and lipids.
Nucleic Acids Res.
PUBLISHED: 10-11-2011
Show Abstract
Hide Abstract
Exosomes are membraneous nanovesicles of endocytic origin released by most cell types from diverse organisms; they play a critical role in cell-cell communication. ExoCarta (http://www.exocarta.org) is a manually curated database of exosomal proteins, RNA and lipids. The database catalogs information from both published and unpublished exosomal studies. The mode of exosomal purification and characterization, the biophysical and molecular properties are listed in the database aiding biomedical scientists in assessing the quality of the exosomal preparation and the corresponding data obtained. Currently, ExoCarta (Version 3.1) contains information on 11,261 protein entries, 2375 mRNA entries and 764 miRNA entries that were obtained from 134 exosomal studies. In addition to the data update, as a new feature, lipids identified in exosomes are added to ExoCarta. We believe that this free web-based community resource will aid researchers in identifying molecular signatures (proteins/RNA/lipids) that are specific to certain tissue/cell type derived exosomes and trigger new exosomal studies.
Related JoVE Video
Identification of Novel Phosphorylation Motifs Through an Integrative Computational and Experimental Analysis of the Human Phosphoproteome.
J Proteomics Bioinform
PUBLISHED: 07-02-2011
Show Abstract
Hide Abstract
Protein phosphorylation occurs in certain sequence/structural contexts that are still incompletely understood. The amino acids surrounding the phosphorylated residues are important in determining the binding of the kinase to the protein sequence. Upon phosphorylation these sequences also determine the binding of certain domains that specifically bind to phosphorylated sequences. Thus far, such motifs have been identified through alignment of a limited number of well identified kinase substrates. RESULTS: Experimentally determined phosphorylation sites from Human Protein Reference Database were used to identify 1,167 novel serine/threonine or tyrosine phosphorylation motifs using a computational approach. We were able to statistically validate a number of these novel motifs based on their enrichment in known phosphopeptides datasets over phosphoserine/threonine/tyrosine peptides in the human proteome. There were 299 novel serine/threonine or tyrosine phosphorylation motifs that were found to be statistically significant. Several of the novel motifs that we identified computationally have subsequently appeared in large datasets of experimentally determined phosphorylation sites since we initiated our analysis. Using a peptide microarray platform, we have experimentally evaluated the ability of casein kinase I to phosphorylate a subset of the novel motifs discovered in this study. Our results demonstrate that it is feasible to identify novel phosphorylation motifs through large phosphorylation datasets. Our study also establishes peptide microarrays as a novel platform for high throughput kinase assays and for the validation of consensus motifs. Finally, this extended catalog of phosphorylation motifs should assist in a systematic study of phosphorylation networks in signal transduction pathways.
Related JoVE Video
Proteomic profiling of secretome and adherent plasma membranes from distinct mammary epithelial cell subpopulations.
Proteomics
PUBLISHED: 03-07-2011
Show Abstract
Hide Abstract
The stem cell niche comprises stem cells (SCs), stromal cells, soluble factors, extracellular matrix constituents and vascular networks. The ability to identify signals that regulate SC self-renewal and differentiation is confounded by the difficulty in isolating pure SC niche components in sufficient quantities to enable their biochemical characterisation. Here, we report the extracellular (secretome) and adherent plasma membrane proteomes of three distinct epithelial cell subpopulations isolated and immortalized from the mouse mammary gland--basal and mammary stem cell (basal/MaSC), luminal progenitor (LP) and mature luminal (ML) cell lines. GeLC-MS/MS-based proteomic profiling revealed a distinct switch in components modulating Wnt and ephrin signalling, and integrin-mediated interactions amongst the three cell subpopulations. For example, expression of ephrin B2, ephrin receptors A1, and A2, as well as integrins ?2?1 and ?6?4 were shown to be enriched in basal/MaSCs, relative to LP and ML cells. Conspicuously, Wnt10a was uniquely detected in basal/MaSCs, and may modulate the canonical Wnt signalling pathway to maintain basal/MaSC activity. By contrast, non-canonical Wnt signalling might be elevated in ML cells, as evidenced by the high expression levels of Wnt5a, Wnt5b, and the transmembrane tyrosine kinase Ror2.
Related JoVE Video
Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database.
Proteomics
PUBLISHED: 02-17-2011
Show Abstract
Hide Abstract
Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell-cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.
Related JoVE Video
Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins.
Methods
PUBLISHED: 01-17-2011
Show Abstract
Hide Abstract
Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC-MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC-MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.
Related JoVE Video
Proteomics profiling of Madin-Darby canine kidney plasma membranes reveals Wnt-5a involvement during oncogenic H-Ras/TGF-beta-mediated epithelial-mesenchymal transition.
Mol. Cell Proteomics
PUBLISHED: 05-28-2010
Show Abstract
Hide Abstract
Epithelial-mesenchymal transition (EMT) describes a process whereby polarized epithelial cells with restricted migration transform into elongated spindle-shaped mesenchymal cells with enhanced motility and invasiveness. Although there are some molecular markers for this process, including the down-regulation of E-cadherin, our understanding of plasma membrane (PM) and associated proteins involved in EMT is limited. To specifically explore molecular alterations occurring at the PM, we used the cationic colloidal silica isolation technique to purify PM fractions from epithelial Madin-Darby canine kidney cells during Ras/TGF-?-mediated EMT. Proteins in the isolated membrane fractions were separated by one-dimensional SDS-PAGE and subjected to nano-LC-MS/MS-based protein identification. In this study, the first membrane protein analysis of an EMT model, we identified 805 proteins and determined their differential expression using label-free spectral counting. These data reveal that Madin-Darby canine kidney cells switch from cadherin-mediated to integrin-mediated adhesion following Ras/TGF-?-mediated EMT. Thus, during the EMT process, E-cadherin, claudin 4, desmoplakin, desmoglein-2, and junctional adhesion molecule A were down-regulated, whereas integrins ?6?1, ?3?1, ?2?1, ?5?1, ?V?1, and ?V?3 along with their extracellular ligands collagens I and V and fibronectin had increased expression levels. Conspicuously, Wnt-5a expression was elevated in cells undergoing EMT, and transient Wnt-5a siRNA silencing attenuated both cell migration and invasion in these cells. Furthermore, Wnt-5a expression suppressed canonical Wnt signaling induced by Wnt-3a. Wnt-5a may act through the planar cell polarity pathway of the non-canonical Wnt signaling pathway as several of the components and modulators (Wnt-5a, -5b, frizzled 6, collagen triple helix repeat-containing protein 1, tyrosine-protein kinase 7, RhoA, Rac, and JNK) were found to be up-regulated during Ras/TGF-?-mediated EMT.
Related JoVE Video
Exosomes: extracellular organelles important in intercellular communication.
J Proteomics
PUBLISHED: 01-29-2010
Show Abstract
Hide Abstract
In addition to intracellular organelles, eukaryotic cells also contain extracellular organelles that are released, or shed, into the microenvironment. These membranous extracellular organelles include exosomes, shedding microvesicles (SMVs) and apoptotic blebs (ABs), many of which exhibit pleiotropic biological functions. Because extracellular organelle terminology is often confounding, with many preparations reported in the literature being mixtures of extracellular vesicles, there is a growing need to clarify nomenclature and to improve purification strategies in order to discriminate the biochemical and functional activities of these moieties. Exosomes are formed by the inward budding of multivesicular bodies (MVBs) and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane (PM). In this review we focus on various strategies for purifying exosomes and discuss their biophysical and biochemical properties. An update on proteomic analysis of exosomes from various cell types and body fluids is provided and host-cell specific proteomic signatures are also discussed. Because the ectodomain of ~42% of exosomal integral membrane proteins are also found in the secretome, these vesicles provide a potential source of serum-based membrane protein biomarkers that are reflective of the host cell. ExoCarta, an exosomal protein and RNA database (http://exocarta.ludwig.edu.au), is described.
Related JoVE Video
NetPath: a public resource of curated signal transduction pathways.
Genome Biol.
PUBLISHED: 01-12-2010
Show Abstract
Hide Abstract
We have developed NetPath as a resource of curated human signaling pathways. As an initial step, NetPath provides detailed maps of a number of immune signaling pathways, which include approximately 1,600 reactions annotated from the literature and more than 2,800 instances of transcriptionally regulated genes - all linked to over 5,500 published articles. We anticipate NetPath to become a consolidated resource for human signaling pathways that should enable systems biology approaches.
Related JoVE Video
Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature.
Mol. Cell Proteomics
PUBLISHED: 10-16-2009
Show Abstract
Hide Abstract
Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.
Related JoVE Video
ExoCarta: A compendium of exosomal proteins and RNA.
Proteomics
PUBLISHED: 10-08-2009
Show Abstract
Hide Abstract
Exosomes, membrane microvesicles (40-100 nm) secreted by most cell types, can be isolated in several ways while characterizing them is heavily based on electron microscopy and, most importantly, the identification of exosome marker proteins. Researchers rely on the identification of certain exosomal marker proteins including Alix, CD9 and CD63 to confirm the presence of exosomes in their preparations. An evolutionary-conserved set of protein molecules have been identified in most exosomes studied to date. However, with the complexity of tissue/cell type-specific proteins being incorporated in the exosomes, some of these so-called exosomal markers are not always present in all the exosomes. The presence of tissue/cell type-specific proteins in exosomes allows researchers to isolate them using immunoaffinity capture methods. A compendium for exosomal proteomes will aid researchers in identifying proteins that were more commonly found in various exosomes (exosome markers) and those that are specific to certain tissue/cell type-derived exosomes. Here, we describe ExoCarta, a compendium for proteins and RNA molecules identified in exosomes. ExoCarta is first of its kind and the resource is freely available to the scientific community through the web (http://exocarta.ludwig.edu.au). We believe that this community resource will be of great biological importance for any future exosome analyses.
Related JoVE Video
Exosomes: proteomic insights and diagnostic potential.
Expert Rev Proteomics
PUBLISHED: 06-04-2009
Show Abstract
Hide Abstract
Exosomes are 40-100-nm diameter membrane vesicles of endocytic origin that are released by most cell types upon fusion of multivesicular bodies with the plasma membrane, presumably as a vehicle for cell-free intercellular communication. While early studies focused on their secretion from diverse cell types in vitro, exosomes have now been identified in body fluids such as urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva and blood. Exosomes have pleiotropic biological functions, including immune response, antigen presentation, intracellular communication and the transfer of RNA and proteins. While they have also been implicated in the transport and propagation of infectious cargo, such as prions, and retroviruses, including HIV, suggesting a role in pathological situations, recent studies suggest that the presence of such infectious cargo may be artefacts of exosome-purification strategies. Improvements in mass spectrometry-based proteomic tools, both hardware and software, coupled with improved purification schemes for exosomes, has allowed more in-depth proteome analyses, contributing immensely to our understanding of the molecular composition of exosomes. Proteomic cataloguing of exosomes from diverse cell types has revealed a common set of membrane and cytosolic proteins, suggesting the evolutionary importance of these membrane particles. Additionally, exosomes express an array of proteins that reflect the originating host cell. Recent findings that exosomes contain inactive forms of both mRNA and microRNA that can be transferred to another cell and be functional in that new environment, have initiated many microRNA profiling studies of exosomes circulating in blood. These studies highlight the potential of exosomal microRNA profiles for use as diagnostic biomarkers of disease through a noninvasive blood test. The exacerbated release of exosomes in tumor cells, as evidenced by their increased levels in blood during the late stage of a disease and their overexpression of certain tumor cell biomarkers, suggests an important role of exosomes in diagnosis and biomarker studies. The aim of this article is to provide a brief overview of exosomes, including methods used to isolate and characterize exosomes. New advances in proteomic methods, and both mass spectrometry hardware and informatics tools will be covered briefly.
Related JoVE Video
Human Protein Reference Database--2009 update.
Nucleic Acids Res.
PUBLISHED: 03-04-2009
Show Abstract
Hide Abstract
Human Protein Reference Database (HPRD--http://www.hprd.org/), initially described in 2003, is a database of curated proteomic information pertaining to human proteins. We have recently added a number of new features in HPRD. These include PhosphoMotif Finder, which allows users to find the presence of over 320 experimentally verified phosphorylation motifs in proteins of interest. Another new feature is a protein distributed annotation system--Human Proteinpedia (http://www.humanproteinpedia.org/)--through which laboratories can submit their data, which is mapped onto protein entries in HPRD. Over 75 laboratories involved in proteomics research have already participated in this effort by submitting data for over 15,000 human proteins. The submitted data includes mass spectrometry and protein microarray-derived data, among other data types. Finally, HPRD is also linked to a compendium of human signaling pathways developed by our group, NetPath (http://www.netpath.org/), which currently contains annotations for several cancer and immune signaling pathways. Since the last update, more than 5500 new protein sequences have been added, making HPRD a comprehensive resource for studying the human proteome.
Related JoVE Video
Human Proteinpedia: a unified discovery resource for proteomics research.
Nucleic Acids Res.
PUBLISHED: 03-04-2009
Show Abstract
Hide Abstract
Sharing proteomic data with the biomedical community through a unified proteomic resource, especially in the context of individual proteins, is a challenging prospect. We have developed a community portal, designated as Human Proteinpedia (http://www.humanproteinpedia.org/), for sharing both unpublished and published human proteomic data through the use of a distributed annotation system designed specifically for this purpose. This system allows laboratories to contribute and maintain protein annotations, which are also mapped to the corresponding proteins through the Human Protein Reference Database (HPRD; http://www.hprd.org/). Thus, it is possible to visualize data pertaining to experimentally validated posttranslational modifications (PTMs), protein isoforms, protein-protein interactions (PPIs), tissue expression, expression in cell lines, subcellular localization and enzyme substrates in the context of individual proteins. With enthusiastic participation of the proteomics community, the past 15 months have witnessed data contributions from more than 75 labs around the world including 2710 distinct experiments, >1.9 million peptides, >4.8 million MS/MS spectra, 150,368 protein expression annotations, 17,410 PTMs, 34,624 PPIs and 2906 subcellular localization annotations. Human Proteinpedia should serve as an integrated platform to store, integrate and disseminate such proteomic data and is inching towards evolving into a unified human proteomics resource.
Related JoVE Video
Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.
PLoS Biol.
Show Abstract
Hide Abstract
Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
Related JoVE Video
Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids.
Mol. Cell Proteomics
Show Abstract
Hide Abstract
Exosomes are naturally occurring biological nanomembranous vesicles (?40 to 100 nm) of endocytic origin that are released from diverse cell types into the extracellular space. They have pleiotropic functions such as antigen presentation and intercellular transfer of protein cargo, mRNA, microRNA, lipids, and oncogenic potential. Here we describe the isolation, via sequential immunocapture using anti-A33- and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from organoids derived from human colon carcinoma cell line LIM1863. The exosome populations (A33-Exos and EpCAM-Exos) could not be distinguished via electron microscopy and contained stereotypical exosome markers such as TSG101, Alix, and HSP70. The salient finding of this study, revealed via gel-based LC-MS/MS, was the exclusive identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3), mucin 13 and the apical intestinal enzyme sucrase isomaltase and increased expression of dipeptidyl peptidase IV and the apically restricted pentaspan membrane glycoprotein prominin 1. In contrast, the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1, the Golgi membrane protein ADP-ribosylation factor, and clathrin. Our observations are consistent with EpCAM- and A33-Exos being released from the apical and basolateral surfaces, respectively, and the EpCAM-Exos proteome profile with widely published stereotypical exosomes. A proteome analysis of LIM1863-derived shed microvesicles (sMVs) was also performed in order to clearly distinguish A33- and EpCAM-Exos from sMVs. Intriguingly, several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos, whereas neither MHC class I nor MHC class II molecules were observed via MS in EpCAM-Exos. Additionally, we report for the first time in any extracellular vesicle study the colocalization of EpCAM, claudin-7, and CD44 in EpCAM-Exos. Given that these molecules are known to complex together to promote tumor progression, further characterization of exosome subpopulations will enable a deeper understanding of their possible role in regulation of the tumor microenvironment.
Related JoVE Video
Identifying mutated proteins secreted by colon cancer cell lines using mass spectrometry.
J Proteomics
Show Abstract
Hide Abstract
Secreted proteins encoded by mutated genes (mutant proteins) are a particularly rich source of biomarkers being not only components of the cancer secretome but also actually implicated in tumorigenesis. One of the challenges of proteomics-driven biomarker discovery research is that the bulk of secreted mutant proteins cannot be identified directly and quantified by mass spectrometry due to the lack of mutated peptide information in extant proteomics databases. Here we identify, using an integrated genomics and proteomics strategy (referred to iMASp - identification of Mutated And Secreted proteins), 112 putative mutated tryptic peptides (corresponding to 57 proteins) in the collective secretomes derived from a panel of 18 human colorectal cancer (CRC) cell lines. Central to this iMASp was the creation of Human Protein Mutant Database (HPMD), against which experimentally-derived secretome peptide spectra were searched. Eight of the identified mutated tryptic peptides were confirmed by RT-PCR and cDNA sequencing of RNA extracted from those CRC cells from which the mutation was identified by mass spectrometry. The iMASp technology promises to improve the link between proteomics and genomic mutation data thereby providing an effective tool for targeting tryptic peptides with mutated amino acids as potential cancer biomarker candidates. This article is part of a Special Issue entitled: Integrated omics.
Related JoVE Video
Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.
Methods
Show Abstract
Hide Abstract
Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.