Dataset Information

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.

SUBMITTER: Clark DJ

PROVIDER: S-EPMC7389820 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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

Clark David J DJ Fondrie William E WE Liao Zhongping Z Hanson Phyllis I PI Fulton Amy A Mao Li L Yang Austin J AJ

Analytical chemistry 20150929 20

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 ex ...[more]

PMID: 26378940

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Project description:Background:Paclitaxel is widely used in the treatment of cancer and has a good effect in the treatment of non-small cell lung cancer. The combination of TMT proteomics and bioinformatics is used to systematically analyze the molecular mechanism of paclitaxel in the treatment of lung adenocarcinoma A549 cell, which is helpful to screen new therapeutic targets. Methods:MTT assay was used to analyze the inhibitory effect of paclitaxel on the proliferation of A549 cells. The proteins were identified by TMT quantitative proteomics and the differential expression proteins (DEPs) database was constructed. The DEPs were enriched by Gene Ontology (GO) and KEGG pathway annotation. Based on the information in the STRING database, find the interaction between DEPs, and the protein-protein interaction (PPI) networks of DEPs were constructed and analyzed by using the Cytoscape software. According to the PPI network results, select the hub proteins from DEPs for WB verification. Results:A total of 5449 proteins were identified in A549 by TMT proteomics. Compared with the control group, 281 DEPs were significantly up-regulated and 218 were significantly down-regulated after paclitaxel treatment. GO functional analysis, we found that the main functions of these DEPs are binding, catalytic activity, molecular function regulator and so on. They are mainly involved in cellular process, metabolic process, biological regulation and so on. KEGG analysis showed that the three most significant signal transduction pathways of DEPs enrichment were DNA replication, steroid biosynthesis, oxidative phosphorylation. In PPI network, there are 294 nodes among which CDK1, MCM2-5 and PCNA are located at the center of proteins interaction. WB analysis confirmed that the expression of CDK1 was significantly down-regulated, consistent with the TMT results. Conclusion:Paclitaxel significantly increased the expression of tubulin, binding tubulin to promote A549 cell death. In addition, paclitaxel significantly inhibited the expression of hub proteins, DNA replication and cell cycle pathways, thus killing lung adenocarcinoma cell A549. These findings will enhance the understanding of the mechanism of paclitaxel in the treatment of lung adenocarcinoma cell A549 and provide new valuable targets.

Dataset Information

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

Publications

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

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