ABSTRACT How does the molecular composition of cells and cell-cell contacts change over time or after experimental manipulations? This fundamental question is asked across all fields of biomedical science and is at the heart of most studies focused on determining cellular and molecular mechanisms of biological phenomenon. Yet, our ability to answer this question is severely constrained by the limitations of current methods to detect the protein composition of particular cellular structures. Imagine the rapid progress biological science could make if we could detect more than a handful of proteins at specified locations on cells in each experiment. The objective of our project is to develop a new approach to achieve this goal?called ?context-rich mass cytometry? ?a technology that enables multi-parametric analysis of proteins in the context of cellular interactions. Mass spectrometry has recently emerged as a powerful tool for cell analysis. One version of this technology called mass cytometry or CyToF is used for multi-parametric analysis of protein expression in single cells. It is similar to flow cytometry in that cells are labeled with antibodies and analyzed on cell-by-cell basis in high- throughput manner. However, unlike flow cytometry which employs fluorescently-labeled antibodies and is limited to ~12 parameters, mass cytometry employs rare earth metals as antibody labels and may be used to analyze up to 60 intracellular or cell surface markers. While an exciting technology, mass cytometry requires that cells be extracted from their native microenvironment and homogenized into single cell suspension prior to analysis. This makes connecting the protein signature to the microenvironment context very challenging. The objective of our project is to develop ?context-rich mass cytometry? ?a technology that enables multi- parametric analysis of proteins in the context of cellular interactions. Once developed this mass spectrometry approach will be used to determine the composition and organization of proteins within immune synapses. This new knowledge enabled by our technology may, in the future, be parlayed into strategies and therapies for preventing infections.
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