Abstract
BackgroundCurrent markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ∼100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery.Materials and MethodsExosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry.ResultsProteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes.ConclusionsIdentification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer.
Highlights
Prostate Specific Antigen (PSA) is a clinically useful protein biomarker for diagnostics and follow-up after treatment for prostate cancer (PCa)
Identification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer
Isolation and characterization Electron Microscopy (EM) of the purified exosome samples revealed that vesicles derived from four cell lines are reasonably homogeneous in size, with an approximate diameter of 70– 200 nm (Figure 1)
Summary
Prostate Specific Antigen (PSA) is a clinically useful protein biomarker for diagnostics and follow-up after treatment for prostate cancer (PCa). In the past few decades, a tremendous amount of research has been performed to find new and better biomarkers for PCa, often using state-of-the-art mass spectrometry technologies, but the discovery of novel low abundance protein has been generally hampered by the complexity of serum or urine [3]. Isolation of exosomes from body fluids represents an attractive approach to bypass these limitations and enable detection of candidate (low abundant) biomarkers. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ,100 nm, which contain proteins that are specific to the tissue from which they are derived and can be considered as treasure chests for disease-specific biomarker discovery
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