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Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. Rare cells in blood and tissue have been shown to serve as specific indicators of disease status and progression, a source of adult stem cells, and a tool for patient stratification and monitoring. Previous reports (1Nagrath S. Sequist L.V. Maheswaran S. Bell D.W. Irimia D. Ulkus L. Smith M.R. Kwak E.L. Digumarthy S. Muzikansky A. Ryan P. Balis U.J. Tompkins R.G. Haber D.A. Toner M. 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Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the cellsearch system.Clin. Cancer Res. 2007; 13: 920-928Crossref PubMed Scopus (1075) Google Scholar), for example, have shown that the concentration of circulating tumor cells (CTCs) within a cancer patient's blood can act as a therapeutic monitoring tool (1Nagrath S. Sequist L.V. Maheswaran S. Bell D.W. Irimia D. Ulkus L. Smith M.R. Kwak E.L. Digumarthy S. Muzikansky A. Ryan P. Balis U.J. Tompkins R.G. Haber D.A. Toner M. Isolation of rare circulating tumor cells in cancer patients by microchip technology.Nature. 2007; 450: 1235-1239Crossref PubMed Scopus (2963) Google Scholar, 2Cristofanilli M. Budd G.T. Ellis M.J. Stopeck A. Matera J. Miller M.C. Reuben J.M. Doyle G.V. Allard W.J. Terstappen L.W. Hayes D.F. Circulating tumor cells, disease progression, and survival in metastatic breast cancer.New Engl. J. 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Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition.Science. 2013; 339: 580-584Crossref PubMed Scopus (1816) Google Scholar). Rare cells in blood or other body fluids represent a particularly challenging problem for discovery proteomic analysis as the volume of the fluid sample is limited and the concentration of cells within that sample is very low. For a blood sample containing rare cells of interest, this low level means capturing a subpopulation of target cells with high recovery and purity from a greatly heterogeneous mixture in only one or a few ml and then performing sample preparation with minimal sample loss. Furthermore, ultra-trace LC-MS needs to be conducted with specially prepared columns with highly sensitive MS, along with advanced data processing. Key to success is the full integration of all the steps in the workflow to achieve the detection level required. The present work combines a series of innovative steps leading to successful discovery proteomic analysis of rare cells. Consider first rare cell isolation for which several approaches have recently been developed (9Pratt E.D. Huang C. Hawkins B.G. Gleghorn J.P. Kirby B.J. Rare cell capture in microfluidic devices.Chem. Eng. Sci. 2011; 66: 1508-1522Crossref PubMed Scopus (161) Google Scholar, 10Zborowski M. Chalmers J.J. Rare cell separation and analysis by magnetic sorting.Anal. Chem. 2011; 83: 8050-8056Crossref PubMed Scopus (147) Google Scholar). A particularly powerful approach is magnet-activated cell sorting (MACS) where antibody-functionalized magnetic beads are utilized to enrich a subset of cells in a complex sample such as whole blood (10Zborowski M. Chalmers J.J. Rare cell separation and analysis by magnetic sorting.Anal. Chem. 2011; 83: 8050-8056Crossref PubMed Scopus (147) Google Scholar, 11Miltenyi S. Müller W. Weichel W. Radbruch A. 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The proteomic of the MCF-7 cells from blood and the from blood very significant in of the is that high or such as and for the For the where the MCF-7 cells in the very to in the analysis of the MCF-7 cells from blood by the microfluidic for and proteins that the high of cell capture the microfluidic platform in isolation of rare cells, first spiked and MCF-7 cells by for all MCF-7 cell in 1 ml of whole blood and using the magnetophoretic of the cells by the and of the cells by concentration and into for The cells as and to of the resulting to analysis in of a sample equivalent to cells in identification of and proteins in in cells spiked in whole blood and cells The of proteomic profiling with the of cells. cells and per injection and in The of the followed a the quantitative spiked in 1 ml of blood MCF-7 cells at concentration levels to cells) in from one to cells, and the two to proteomic profiling in where of isolate in analysis and cells to the using the approach B. D. J. J. 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