Breast cancer exosome protein profiling using TMT quantitation and SVM analysis

ABSTRACT: 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 onco-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 10,000 x g cellular debris pellet, a 100,000 x g crude exosome pellet, and an Optiprep enriched exosome pellet. In our TMT analysis, we identified and quantified 2,179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in our exosome fraction based on our TMT protein ratios. Employing SVM cluster analysis allowed us to classify 241 proteins as “true” exosomal cargo proteins. Annotation tools revealed breast cancer derived exosomes were enriched in plasma membrane proteins with functional roles in cell adhesion and cell immunity, in addition to several cell signaling components. This study provides a robust and vigorous framework for the future development of using exosomes as potential multi-protein marker phenotyping tool in breast cancer diagnosis.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Breast Cancer Cell Line

DISEASE(S): Breast Cancer

SUBMITTER: David Clark  

LAB HEAD: Austin Yang

PROVIDER: PXD002053 | Pride | 2015-10-02

REPOSITORIES: Pride