Proteomics Analysis of Bladder Cancer Exosomes
Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. Bladder cancer is one of the eight most frequent cancers in the Western world, and the frequency of transitional cell carcinoma (TCC), 1The abbreviations used are:TCCtransitional cell carcinoma5T4trophoblast glycoproteinBCAbicinchoninic acidFDRfalse discovery rateHLAhuman leukocyte antigenhsp90heat shock protein 90LAMPlysosome-associated membrane proteinMHCmajor histocompatability complexTEABtriethylammonium bicarbonateTSG101tumor susceptibility gene 101NHSNational Health ServiceFBSfetal bovine serumRTroom temperatureBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolhnRNPheterogeneous nuclear ribonucleoprotein2DEtwo-dimensional electrophoresisIDidentifierORAoverrepresentation analysis.1The abbreviations used are:TCCtransitional cell carcinoma5T4trophoblast glycoproteinBCAbicinchoninic acidFDRfalse discovery rateHLAhuman leukocyte antigenhsp90heat shock protein 90LAMPlysosome-associated membrane proteinMHCmajor histocompatability complexTEABtriethylammonium bicarbonateTSG101tumor susceptibility gene 101NHSNational Health ServiceFBSfetal bovine serumRTroom temperatureBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolhnRNPheterogeneous nuclear ribonucleoprotein2DEtwo-dimensional electrophoresisIDidentifierORAoverrepresentation analysis. which accounts for 90% of bladder cancers, is second only to prostate cancer as a malignancy of the genitourinary tract. Urine cytology and cystoscopy remain the predominant clinical tools for diagnosing and monitoring the disease, but cytology is poorly sensitive, particularly for low grade tumors, and does not serve as a prognostic tool. Cystoscopy is an invasive procedure, and there is pressing need to that used to transitional cell carcinoma trophoblast glycoprotein discovery human leukocyte antigen shock protein membrane protein major histocompatability complex susceptibility gene Health bovine nuclear analysis. transitional cell carcinoma trophoblast glycoprotein discovery human leukocyte antigen shock protein membrane protein major histocompatability complex susceptibility gene Health bovine nuclear analysis. vesicles exosomes that are present in fluids vesicles are present in human and of exosomes in human Exosomes with are present in human and of analysis of exosomes from human have been as a of and of exosomes in human of exosomes as of identified by a urinary biomarker for urine a to for prostate These nanometer-sized vesicles, which are secreted by most cell types, from of the and a of the Exosomes are in membrane and and this to of particular to the vesicles as of Exosomes also an of and which are in of and is a of exosomal are not present as in as which are the exosome of exosomes as of of and is a of the to exosomes from urine and of exosomes in human vesicles are present in human vesicles with in human other Exosomes with are present in human for and complex of in a non-invasive analysis may of in disease diagnosis and monitoring in a of of exosomes as of identified by a urinary biomarker for a for exosomes and their of cell of proteins with Exosomes as of were first in the context of a proteomics revealed in urinary exosome identified by a urinary biomarker for The identified as a that in was also in with before in urinary were identified by a urinary biomarker for exosome analysis may also for cancers, as cancer, the of cell exosomes may with exosomes not in of exosomes as of a for in this there is and disease of exosomes as of a for studies of urinary exosomes in prostate cancer with exosomes protein urinary exosomes as in prostate prostate cancer gene urine a to for prostate urine a to for prostate of prostate cancer with prostate exosomes have not been studied in the context of other urological as cancer, and to only one report the from bladder cancer and of urinary of bladder In that the proteome of a highly complex of and other urinary that by high eight proteins that may in the of the is proteins are of the and most in and other clinical a major In this is exosomes present complex fluids from cell exosomes may from cells vesicles are present in human and a may from as the and proteome of exosomes secreted by and other as of prostate cancer exosomes present in urine from cells of the and of the and of exosomes in human urine a to for prostate urinary exosomes as in prostate proteomics studies of exosomes from fluids are by the of high proteins the and present in exosomes from analysis of exosomes from human and protein present in exosomes purified from urine and of exosomes in human in the of the data by proteomics requiring of the proteins of The protein of exosomes using a cell is one that may used to the protein of exosomes by various cell remain major in the of exosome proteomics that of of identified proteins. are the for exosome that in studies have to contaminants a of the vesicles, their to on sucrose and of clinical grade exosomes from other and of have a and approach. 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