Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios.
Journal of extracellular vesicles. 2016 Jun 29*** epublish ***
Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton
Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom., Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom., Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom., Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom., Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom., Metabolomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Derio, Spain., Metabolomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Derio, Spain., Department of Computer Science, College of Science, Swansea University, United Kingdom., Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom., Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom., Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom.