TY - JOUR
T1 - Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis
AU - Clark, David J.
AU - Fondrie, William E.
AU - Liao, Zhongping
AU - Hanson, Phyllis I.
AU - Fulton, Amy
AU - Mao, Li
AU - Yang, Austin J.
N1 - Publisher Copyright:
© 2015 American Chemical Society.
PY - 2015/10/20
Y1 - 2015/10/20
N2 - 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.
AB - 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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U2 - 10.1021/acs.analchem.5b02586
DO - 10.1021/acs.analchem.5b02586
M3 - Article
C2 - 26378940
AN - SCOPUS:84945318776
VL - 87
SP - 10462
EP - 10469
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 20
ER -