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. 2014 Apr;13(4):1050-64.
doi: 10.1074/mcp.M113.032136. Epub 2014 Feb 6.

Proteomics analysis of cancer exosomes using a novel modified aptamer-based array (SOMAscan™) platform

Jason Webber  1 , Timothy C Stone, Evaldas Katilius, Breanna C Smith, Bridget Gordon, Malcolm D Mason, Zsuzsanna Tabi, Ian A Brewis, Aled Clayton
Affiliations

Proteomics analysis of cancer exosomes using a novel modified aptamer-based array (SOMAscan™) platform

Jason Webber et al. Mol Cell Proteomics. 2014 Apr.

Abstract

We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes. The technology uses a new class of protein binding reagents called SOMAmers® (slow off-rate modified aptamers) and allows the simultaneous precise measurement of over 1000 proteins. Exosomes were highly purified from the Du145 prostate cancer cell line, by pooling selected fractions from a continuous sucrose gradient (within the density range of 1.1 to 1.2 g/ml), and examined under standard conditions or with additional detergent treatment by the SOMAscan™ array (version 3.0). Lysates of Du145 cells were also prepared, and the profiles were compared. Housekeeping proteins such as cyclophilin-A, LDH, and Hsp70 were present in exosomes, and we identified almost 100 proteins that were enriched in exosomes relative to cells. These included proteins of known association with cancer exosomes such as MFG-E8, integrins, and MET, and also those less widely reported as exosomally associated, such as ROR1 and ITIH4. Several proteins with no previously known exosomal association were confirmed as exosomally expressed in experiments using individual SOMAmer® reagents or antibodies in micro-plate assays. Western blotting confirmed the SOMAscan™-identified enrichment of exosomal NOTCH-3, L1CAM, RAC1, and ADAM9. In conclusion, we describe here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmer®-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings.

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Conflict of interest statement

Conflict of interest statement: Evaldas Katilius, Breanna C. Smith and Bridget Gordon are employed by SomaLogic Inc.

Figures

Fig. 1.
Fig. 1.
Preparation of highly pure Du145 exosomes for analysis via SOMAscanTM. Du145 exosomal vesicles were separated on a continuous sucrose gradient, and the density of 15 collected fractions was determined. Nanoparticle tracking analysis was performed on each fraction, and the particle concentration was plotted against the fraction density. Bars represent mean ± S.D. of duplicate measurements (A). The size distribution of particles within each fraction is shown, and the density of each fraction is specified, revealing single-peak, monodisperse populations of small vesicles in fractions of classical exosomal density (between 1.1 and 1.2 g/ml) (B). A proportion of each fraction was coated onto latex microbeads, stained with antibodies as specified, and analyzed via flow cytometry. Bars represent median fluorescence values from 5000 events, and the positions of fractions 8–10 are annotated (C). This characterization aided in selecting relevant fractions—specifically, F8, F9, and F10—that were pooled for subsequent array analyses.
Fig. 2.
Fig. 2.
Comparison of exosomes and cells under SB17 + Nonidet P-40/DCO conditions. Exosome and cell preparations under SB17 + Nonidet P-40/DCO conditions were compared and the data were filtered to eliminate poor replicates. Data are represented as a heat map highlighting the comparison between exosomes and their parent cells (A). The data identified 57 proteins that were elevated 2-fold or greater in exosomes relative to cells, and some examples of these are shown (bars show mean RFU values ± S.E. of triplicates, and the p value is shown) (B). Eight proteins exhibited comparable expression levels (of less than 0.1-fold change) in exosomes and cells (C), whereas 89 exhibited >2-fold elevated expression in cells (D). The analytes displaying >2-fold elevation in exosomes and those with an RFU signal of >200 units are shown (E), plotted as fold enrichment (left axis) and RFU values (right axis). A simple multiplication of fold-increase × log2(RFU), used as a means of identifying proteins that may be both enriched and highly abundant in exosomes, is shown (F). Some candidate proteins were selected from this plot for subsequent validation analyses, and these are indicated with †.
Fig. 3.
Fig. 3.
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 1.5-fold or greater increased expression in exosomes relative to cells, and some examples of these are shown (bars show mean RFU ± S.E. of triplicates, with specified p values) (B). 24 proteins exhibited comparable expression levels (of less than 0.1-fold difference) (C), whereas 574 proteins were >1.5-fold more abundant in cells (D). The data were filtered to include only analytes reporting >1.5-fold elevation in exosomes and those with an RFU signal of >200 units, and these are shown (E), plotted according to fold enrichment (left axis) and RFU values (right axis). A simple multiplication of fold-increase × log2(RFU), used as a means of identifying proteins that may be both enriched and highly abundant in exosomes, is shown (F). Some candidate proteins were selected from this plot for subsequent validation analyses, and these are indicated with †.
Fig. 4.
Fig. 4.
Confirmation of the expression of selected SOMAscanTM-identified proteins. Du145 exosomes purified via the sucrose cushion method were immobilized at specified doses on ELISA microplates and probed using individual SOMAmers® (A) or an indirect staining method with antibodies (B). Signals were detected using Europium-streptavidin and time-resolved fluorimetry (TRF) in each case (bars represent mean ± S.E. of duplicate measurements). SOMAmers® (KDGL or Spuriomer) act as irrelevant controls for nonspecific binding (A). 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 1.1 and 1.2 g/ml are annotated (C). Whole cell lysates and exosomes normalized for protein were subjected to SDS-PAGE and Western blotting and probed with antibodies as indicated. 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. After washing, signals were detected using Europium-streptavidin and TRF (bars represent mean ± S.E. of quadruplicate measurements) (E).
Fig. 5.
Fig. 5.
Biological themes related to exosome-enriched proteins. Network diagrams showing the relationship of biological themes that were significantly enriched in the list of 50 genes with the greatest fold change in exosomes relative to cells for the SB17 + Nonidet P-40/DCO conditions (A) and the SB17 conditions (B). Edge thickness indicates the number of genes shared between terms. Key: GO, Gene Ontology; IPR, InterPro; SM, smart domains; the remaining terms are UniProt derived. The DAVID terms used to derive these diagrams can be found in supplemental Table S4.
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