Abstract
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.
Highlights
From the ‡Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts; ¶Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts; §Department of Chemical Engineering, Northeastern University, Boston, Massachusetts
We have shown that reduction of the LC column diameter in a high resolution porous layer open tube (PLOT)1 format utilizing ultra-low flow can generate a significant gain in limited sample proteomic profiling capabilities [22]
MS data were processed to enable quantitative proteomic profiling using both label-free and isotope reference-based techniques followed by gene ontology analysis
Summary
Rare cell isolation is followed by a series of sample preparation steps, for example cell lysis and protein extraction and digestion. To assess the performance of the microSPE-PLOTnLC-MS platform in proteomic profiling of a limited number of cells, we prepared a lysate of 10 million human MCF-7 cells using conventional lysis and trypsin digestion techniques (see Supplemental Materials).
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