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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M600465-MCP200v1 6/11/1997    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kulasingam, V. Articles by Diamandis, E. P. Search for Related Content PubMed PubMed Citation Articles by Kulasingam, V. Articles by Diamandis, E. P.

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Research

Proteomics Analysis of Conditioned Media from Three Breast Cancer Cell Lines

A Mine for Biomarkers and Therapeutic Targets^*,S

Vathany Kulasingam^, and Eleftherios P. Diamandis^,¶,||

From the Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and ^¶ Department of Clinical Biochemistry, University Health Network and Toronto Medical Laboratories, Toronto, Ontario M5G 1X5, Canada

A "bottom-up" proteomics approach and a two-dimensional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 µg/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers.

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