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Anal Chem. 2015 Oct 20;87(20):10462-9. doi: 10.1021/acs.analchem.5b02586. Epub 2015 Sep 29.

Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis.

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Department of Oncology and Diagnostic Sciences, University of Maryland School of Dentistry , Baltimore, Maryland 21201, United States.
Marlene and Stewart Greenebaum Cancer Center, University of Maryland , Baltimore, Maryland 21201, United States.
Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.
Lily Research Laboratory, Eli Lily and Company , Indianapolis, Indiana 46285, United States.
Department of Cell Biology and Physiology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.


Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as "true" exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression.

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