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

Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes

“…Comparing the molecular signatures of tumor- (26)(27)(28)(29) and DC-derived (our work) medium EVs will surely provide additional specific markers. So far, we have not identified a protein that would be exclusively in medium EVs, and not also in the largest ones, which the other groups working on tumorderived EVs did not evaluate: although it would be very useful, finding a specific marker for these particular EVs may be challenging.…”

“…To obtain these results, it was necessary to combine several separation procedures and to follow a nonbiased approach, rather than a molecule-or EV subtype-targeted one. Some groups have recently performed comparative proteomic analyses of medium-sized and small EVs (with or without floatation into a gradient) from platelets (19) or tumor cells (26)(27)(28)(29). Another group has compared the expression of a few surface proteins on B lymphoma-EVs immunoprecipitated with either CD9, CD63, or CD81 (30).…”

Quantitative Proteomic Analysis of Serum Exosomes from Patients with Locally Advanced Pancreatic Cancer Undergoing Chemoradiotherapy

“…20 Tumor-derived exosomes can be isolated from patients using a number of different techniques including affinity enrichment and ultracentrifugation and thus can provide a minimally invasive method for therapeutic monitoring. 21–24 …”

Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically Relevant Samples

“…There is, however, yet to be a consensus on the methodological details of SEC. For example, various column types have been used, including Sepharose CL‐2B, Sepharose CL‐4B, Sephacryl S‐400, and pre‐packed commercially available columns . This methodological variability makes it difficult to ascertain the true utility of this method for preparing EVs suitable for proteomic analysis.…”