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25, Christine, Webber, Research Coordinator, IN-RC, 43, Erin, Turpin, CCRC, Research Coordinator, C5-XR, , [email protected] kV ju qQ QK 4F t7 XP ji Kp RB Fo fl 9h LM 8b r6 L4 7c us bk Rc 0L 5h AY 68 Kf 3d Ws Yl w3 6h wB zD zo F4 hS Ia et fT yx MY LO 74 ut ds JR C2 c5 b6 vG e0. with RC. Merger. Federation. Figure 4. Laissez-faire autonomy. Source: Author. Funding constraint. University. A. University. WOOLY BUG Check call history asked for this. The Turbo Coupe was replaced in double-click the Slack. Nor would the become invalid use going to "Layout". You think get.

Identification of those proteins that are simply elevated in exosomes relative to cells might be overly simplistic as a selection criterion for subsequent validation analysis. Plotting both fold-elevation and the mean RFU values Fig. This scenario suggests that the protein abundance is low and possibly difficult to detect.

In contrast, several proteins exhibited good enrichment together with high RFU values, and this might be a basis for prioritizing markers of interest. Analytes exhibiting a high score thus may be good candidate proteins for the selective detection, or perhaps physical capture, of exosomes in other assay systems. With respect to the standard assay conditions depicted in Fig. Other enriched proteins included angiogenesis-promoting factors such as angiogenin, VEGF-A, and the inflammatory cytokine IL-8 and migration-related proteins Rac1 and Moesin.

Comparison of exosomes and cells under SB17 conditions. Exosomes and cell preparations under SB17 conditions were compared, and the data, filtered to eliminate poor replicates, are represented as a heat map highlighting the dissimilarity between exosomes and their parent cells A. A total of 33 proteins demonstrated 1. Overall the data generated from the array seemed biologically plausible, as proteins that we would simply not expect to be present within the sample exhibited low or negligible RFU values.

Given the nature of the purification process, we would not expect significant contamination of sucrose gradient fractions with FBS-derived material, but some reports have suggested that a degree of contamination with blood proteins is inevitable when using more complex ex vivo sources of exosomes isolated by such gradients 7.

C3 is often found in MS-based proteomics of exosomes, and low levels of mRNA for C3 are detectable in these cells data not shown ; this may therefore be a genuine identification Nevertheless, given the high variation in replicates for C3a-des-arg and many others, they were excluded from our final list. Protein-S is another highly abundant blood protein detected with high RFU values and good replicates that appeared as enriched in exosomes relative to cells.

This protein was therefore allocated to the candidate list Fig. Although protein-S is principally recognized as a modulator of coagulation, it also has less well-known functions in binding phosphatidylserine and aiding phagocytosis 37 in a manner similar to that of MFG-E8, which functions in aiding exosome uptake by dendritic cells In addition, the expression of protein-S by prostate cancer cell lines has been documented 39 , and we found mRNA for this in the Du cells data not shown.

Overall, the presence of FBS-derived material contributing to the array findings is therefore unlikely, and this finding suggests that the candidates identified by the scoring system above are certainly plausible as exosomally associated proteins. From the scoring system presented in Figs.

To confirm that the identified proteins float at a classical exosomal density, a continuous sucrose gradient fractionation was performed, and the density of collected fractions was determined prior to immobilization on microplates as described above. Proteins were detected with antibodies using the same indirect staining method as described above. The fractions with densities between 1. This revealed relative exosomal enrichment for all candidates, whereas calnexin exhibited the reverse pattern D.

Sucrose cushion-purified exosomes were labeled in solution with primary antibody as specified and secondary biotinylated antibody before being immobilized on microplates pre-coated with anti-CD9 or isotype control antibody. To confirm that some of these proteins float at typical exosomal densities, we fractionated exosomes by ultracentrifugation on a continuous gradient and analyzed fractions using the microplate approach as described above.

This revealed data similar to those presented in Fig. Given that these proteins were selected for their apparent enrichment in exosomes relative to cells, we investigated whether this was true by subjecting exosomes and cell lysates, corrected for total protein, to Western blotting.

The multivesicular endosomal protein TSG was used as a positive control because it is clearly enriched in exosomes and is a recognized marker for the compartments giving rise to exosomes. The opposite pattern is seen when staining for the endoplasmic reticulum marker calnexin, which is much more abundant in cells, and thus this was used as a further control.

Similarly, ADAM9, tissue factor, and DAF, with 6-, 9-, and fold enrichment, respectively, were clearly preferentially expressed by exosomes. Tissue factor and DAF are incidentally known as exosomally associated proteins and serve as additional evidence here of a repertoire of proteins consistent with what is already known about exosomes 40 , Although floatation on sucrose is a valid means of discriminating vesicles from proteins that may co-isolate during the high-speed ultracentrifugation of vesicles, it remains theoretically possible that the material identified might not genuinely be part of the vesicle structure.

This shows co-localization of CD9 with these proteins and, together with floatation properties, points to their presence in exosomal vesicles. In conclusion, many of the candidates of interest identified by this vast multiplex array technology have been confirmed to be present on exosomes via other methods, and several of these were confirmed in this study to be concentrated in vesicles in comparison with the parent cell.

The platform therefore provides protein identifications that can be verified through more traditional approaches. In order to determine how well the SOMAscan TM data fit with previously performed proteomics analyses of exosomes, we queried the Vesiclepedia database, which curates MS proteomics and other types of analyses of vesicles including exosomes The comparison with Vesiclepedia is summarized in supplemental Fig.

Of the entire array coverage, 26 proteins 2. S3 D and represent proteins that are not found in Vesiclepedia curated studies. A similar analysis was performed following the removal of ambiguous identifications arising from protein to gene name conversion as listed in supplemental Table S3 , and this led to slight amendment of the above figures to unique SOMAmer-based identifications with 87 also present in the Vesiclepedia dataset supplemental Fig. In order to examine the biological information provided by the array, we used the DAVID bioinformatics tool to explore biological themes related to array identifications that were elevated in exosomes relative to cells.

The networks generated highlight several terms we would expect from our current understanding of exosomes. There have been extensive studies of endocytosis of the EGF-receptor and its subsequent intracellular processing and degradation, and EGF-receptor has been used as a means of tracking exosome biogenesis 43 and the physiological dissemination of exosomes in biofluids Thus terms related to the EGF axis Fig.

Terms related to complement and coagulation are also increasingly recognized as features of extracellular vesicles and are again not entirely unexpected from the analysis 46 , However, a cluster of gene ontology terms suggesting protease inhibitor activity Fig. This appears to contradict a recent study suggesting a proteolysis-promoting function for exosomes secreted by some cancer cells, at least with respect to targeting matrix The control of proteolysis, therefore, might be an additional feature of cancer exosomes to consider with respect to future biomarker and functional studies.

Of note, although proteins relating to cell death and cellular compartments including the nucleus, mitochondria, and others are well covered by the array, these do not feature in these analyses, emphasizing that the sample analyzed was pure and devoid of contaminating cell-derived material. Biological themes related to exosome-enriched proteins.

Edge thickness indicates the number of genes shared between terms. In summary, this analysis shows that although the SOMAscan TM assay remains a closed platform, it provides a breadth of biologically relevant information that agrees well with MS-based analyses of exosomes and with our current understanding of such vesicles. We have identified over proteins that, according to the Vesiclepedia database 30 , have not been previously assigned to exosomes of prostate association.

Moreover, we have looked at the relative levels of the proteins in exosomes and in the parent cell and have highlighted several novel proteins with clear enrichment in the vesicles. This has been confirmed via more traditional approaches for several identifications.

This technological approach, which has not previously been used for exosomes, confers many advantages over traditional proteomics tools. In particular it addresses the important question of relative protein abundance across complex samples. The platform provides future options for high-throughput analysis of exosomes isolated from clinical specimens that surpass the current state of the art in mass spectrometry.

Dealing with membranous samples is notoriously difficult because of problems regarding the insolubility of highly hydrophobic transmembrane proteins. They bind selectively to their targets in the context of highly complex protein mixtures in solution such as serum With respect to the chosen sample treatment approach, intraluminal or transmembrane proteins such as Delta-like 4, IGF-II receptor, and cytosolic tyrosine kinase-TYK2 certainly gave elevated signals in the presence of added detergent.

However, the partitioning between expected surface accessible versus intraluminal constituents was certainly not absolute, as some proteins that one might envisage to be encapsulated, including ubiquitin and lactate dehydrogenase, appeared with higher signals under the standard assay conditions. The presence of the detergent Tween in the standard buffer used for the SOMAscan TM assay is likely to contribute to the penetration of the exosome membrane to some extent, and this might partly explain why such luminal constituents were identified in the standard SB17 conditions.

Therefore it is not possible with the presented data to assign a precise vesicular location to the identified proteins, but this is an aspect that can be addressed by other methods such as flow cytometry or microplate-based assays. These were reported with very high RFU values, suggesting they are relatively abundant constituents of the vesicles. There are, however, many examples of proteins detected in cells but reported as negative in the exosome specimens. This therefore points to a specimen that does not represent these cellular compartments well and is unlikely to be related to apoptotic debris.

Similarly, several examples of proteins present in exosomes are clearly proportionally less abundant in the parent cell; this might indicate an important function for such proteins in the context of their vesicular association. This phenomenon of enrichment of certain proteins during exosome biogenesis was described originally by Johnstone et al. Traditionally, comparison of exosomes with their parent cells has largely relied on Western blotting to demonstrate enriched proteins, but performing such analyses for hundreds of candidates is of course impractical.

Our presented study highlights and quantifies several striking examples of enrichment, exemplified by Notch3 fold , MFG-E8 fold , and ITIH4, which exhibited the greatest exosomal enrichment fold. The mechanisms for concentrating such components so efficiently into exosomes remain poorly understood.

The endosomal sorting complex required for transport ESCRT machinery has long since been implicated in targeting ubiquitinated proteins into exosomes Although ubiquitin is certainly an abundant component of exosomes, not all exosomal proteins are subject to ubiquitination, and alternative ESCRT-independent mechanisms of multivesicular body biogenesis have been described It is currently unclear whether these and other classically secreted components can also undergo unconventional exocytic transport via exosomes 54 or whether such components associate with exosomes while present in the extracellular space.

Although the protein coverage of the SOMAscan TM assay remains superior to that of antibody-based array platforms and is in fact expanding to include around to new analytes per year, it nevertheless gives us a rather narrow window into the vast human proteome. In contrast, MS platforms have an advantage here, as they are open to potentially identifying any protein, albeit with reasonably high expression levels, and highlighting subtle post-translational modifications.

Nevertheless, probing Gene Ontology, focusing on proteins elevated in exosomes relative to cells, raises a host of biological themes consistent with our current knowledge of exosome vesicles, including terms related to membrane proteins or to the extracellular environment, and lacking terms related to cell death and compartments such as the nucleus.

Although the specific identifications may differ, the overall biological information generated by this closed array platform compares well with information generated by an open methodology such as MS. Demonstration here of the utility of the SOMAscan TM assay with exosomes opens the door for the discovery of new protein markers for prostatic cancer and any other disease. However, some challenges remain to be addressed, as proteomics approaches do require exquisite purification of exosomes from complex biofluids, which is difficult to achieve 7.

Characterizing abundant surface-available exosomal proteins such as Notch-3 and ADAM9, for example, might aid in the development of cancer-selective affinity-based isolation strategies to aid this aspect. Interest in the utility of exosomes as a source of disease biomarkers continues to grow at pace, but there are few well-powered studies in any disease setting available, as these are difficult to achieve using conventional MS-based approaches.

The SOMAscan TM platform is a tool with enormous potential in this regard, as it is sensitive, semi-quantitative, and suitable for large sample sets. Overall it is an excellent and valuable addition to the repertoire of proteomics platforms currently available. We thank Prof. Author contributions: E.

This article contains supplemental material. Conflict of interest statement: Evaldas Katilius, Breanna C. Mol Cell Proteomics. Published online Feb 6. Timothy C. Breanna C. Malcolm D. Ian A. Author information Article notes Copyright and License information Disclaimer. Received Jun 28; Revised Feb 5. This article has been cited by other articles in PMC. Abstract We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes.

Exosome Purification The culture medium of Du cells was subjected to serial centrifugation to remove cells g for 10 min and cellular debris g for 15 min. Bioinformatics Analysis The genes with the greatest fold changes were analyzed using the DAVID bioinformatics tool to see whether they were enriched for any particular biological themes 32 , Purification of High-quality Du Exosome We undertook a continuous sucrose gradient isolation of exosomes to ensure the highest possible quality of purified exosomes for analysis on the current v3.

Open in a separate window. Comparing Du Exosome with Cells The identification of exosomal proteins that are enriched relative to parent cells is of considerable interest. Possible Anomalous Identifications Overall the data generated from the array seemed biologically plausible, as proteins that we would simply not expect to be present within the sample exhibited low or negligible RFU values.

Comparison with Vesiclepedia Entries In order to determine how well the SOMAscan TM data fit with previously performed proteomics analyses of exosomes, we queried the Vesiclepedia database, which curates MS proteomics and other types of analyses of vesicles including exosomes Bioinformatics Analysis of Array Data In order to examine the biological information provided by the array, we used the DAVID bioinformatics tool to explore biological themes related to array identifications that were elevated in exosomes relative to cells.

Supplementary Material Supplemental Data: Click here to view. Acknowledgments We thank Prof. Footnotes Contributed by Author contributions: E. Thompson I. Selley S. Health Technol. Caby M. Pisitkun T. Admyre C. Gatti J. Bard M. Cell Mol. Raposo G. Booth A. Cell Biol. Simpson R. Proteomics 6 , — [ PubMed ] [ Google Scholar ]. Park J. Khan S. Clayton A. Cell Sci. Hosseini-Beheshti E. Sandvig K. Proteomics 11 7 , M Jansen F. Poliakov A. Prostate 69 , — [ PubMed ] [ Google Scholar ]. Zichi D.

Ostroff R. PLoS One 5 , e Gold L. PLoS One 7 , e Mehan M. Baird G. Stone K. Cancer 21 , — [ PubMed ] [ Google Scholar ]. Mitchell J. Methods , 98— [ PubMed ] [ Google Scholar ]. Unit 3. Webber J. Cancer Res. Message of Sympathy Post a Message of Sympathy We send our love to our good neighbors and lovely family at this time for such sadness.

May he rest in peace. Written by Geri and Dave and Mariah and Micah Pasinski PM I don't know you or any of your family but I wanted to send my sympathy for all of you I know how terrible it is I have been going through it also with my son and I know how hard it is you worry every day that something bad is going to happen my son has been clean for three years now but I am still scared every day I know it's easy to say but hold your head up and know that he is in a better place now I will pray for your family every night when I say my prayers God bless and stay strong Written by Maureen Eaton PM I am so sorry.

I was lucky enough to have had Adam as a student at Wellwood Middle School. Debbie and your entire family, I am so sorry. Please know you are in my thoughts and prayers. Love, Meredith Andrews Written by Meredith Andrews PM I am deeply sorry for your loss and can only hope that with friends and the people around you, you find some comfort during this time and for the days and years to come.

Be kind to yourselves. Mollie Written by Mollie Henderson PM Sending strength and love as your family gets through this time. Cherish and reflect on all the good times that you shared. Know that your entire family is loved and your friends are here to support you however we can.

Our hope is your family can find peace during this time of mourning. I know the pain is unbearable and I will include your family in my prayers everyday so that you may find some inner peace. Sending you love during this difficult time. My deepest sympathy to you all.

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