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 SOMAscanTM 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 SOMAscanTM-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.
Highlights
IntroductionHere over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmerா-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings
From the ‡Institute of Cancer and Genetics, School of Medicine, Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff CF14 2TL, United Kingdom; ¶Central Biotechnology Services and Institute of Translation, Innovation, Methodology and Engagement, Henry Wellcome Building, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, United Kingdom; ʈInstitute of Psychological Medicine and Clinical Neurosciences, School of Medicine, Cardiff University, Hadyn Ellis Building, Maindy Road, Cardiff CF24 4HQ, United Kingdom; **SomaLogic Inc, 2945 Wilderness Pl, Boulder, CO 80301
Compatibility of Exosomes with the SOMAscanTM Array Platform—It was important to investigate various sample preparation conditions to maximize the signal for as many analytes as possible, as exosomes were a hitherto untested specimen type for the SOMAscanTM array system
Summary
Here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmerா-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings. Prostate carcinoma is the most frequent male cancer, with an estimated 240,000 newly diagnosed individuals and 28,000 deaths in the United States during 2012 (National Cancer Institute (NIH)). Methods for detecting this cancer are based on a combination of physical examination through digital rectal examination, clinical imaging, quantification of circulating levels of prostate specific antigen (PSA), and transrectal ultrasound-guided biopsy. Exosomes are enriched in membrane proteins and in factors related to such endosomal compartments. Exosomes from diseased origins can be distinguished from those of a normal phenotype based on their protein profiles alone
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