Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis - PubMed (original) (raw)

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

David J Clark et al. Anal Chem. 2015.

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.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.

Figure 1.

Overview of exosome isolation, characterization, and TMT-labeling strategy to elucidate exosomal protein enrichment. (A) Differential ultracentrifugation was performed, and pellets obtained after 10 000g spin (10K pellet), 100 000g spin (100 K pellet), and post-Optiprep density gradient (Opti pellet) were retained for MS analysis. (B) Immunoblot for known exosome markers ALIX and CD63 in the recovered 1 mL Optiprep fractions. Fractions 6 and 7 were positive for the exosome markers with a corresponding density of 1.14–1.19 g/mL. (C) Electron microscopy image of negatively stained SKBR3B exosome vesicles. Bar = 100 nm. (D) Peptide digest from each fraction was TMT-labeled and mixed equally prior to LC–MS/MS analysis. Hybridization method of PQD-CID allowed for quantification of reporter ions and identification of the peptide.

Figure 2.

Figure 2.

Peptides and proteins display differential abundance in various analysis fractions. Identified peptides from ATP synthase subunit δ (A) and CD63 (B) display differences in TMT reporter ion intensities between the three fractions. TMT reporter ions are as follows: 129–10K fraction, 130–100 K fraction, and 131-Opti fraction. (C) Western Blot analysis of individual fraction lysates reveals differential protein abundance (left). Known exosome proteins ALIX, TSG101, and CD63 as well as GSK3_β_ increased in abundance with exosome enrichment. Levels of nuclear proteins (PARP1 and Histone H3 (HISTH3A)), cytoskeletal protein _β_-actin, and the mitochondrial protein, Cytochrome C (CYC1), decreased during exosome enrichment. Reported QuantiMORE TMT ratios for each respective protein representing 100 000g ultracentrifugation (100K/10K) and Optiprep (Opti/10K) exosome enrichment (right).

Figure 3.

Figure 3.

Known exosome markers localize to the quadrant corresponding to 100 K and Optiprep enrichment. TMT log2 protein ratios were plotted by 100K enrichment (x-axis) against Optiprep enrichment (y-axis). Selected Exosome (red) and Nonexosome (blue) protein markers are annotated.

Figure 4.

Figure 4.

SVM cluster analysis classified 241 proteins as exosomal: (A) plotted SVM cluster of all 2 179 quantified proteins from the TMT analysis. Exosome proteins (red circles) localized in the upper right quadrant, with exosome markers and nonexosome markers indicated by black circles. Proteins classified as nonexosomal are indicated by the blue squares. (B) Plotted SVM cluster with annotated plasma membrane markers indicated by black circles.

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