Proteome Capture in Hydrogel Beads for High Resolution Single Cell Analysis

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Summary Analysis of surface and intracellular proteins at the single cell level has become widespread in immunology, oncology, and diagnostics. Although several platforms have emerged for single cell analysis, flow cytometry has remained one of the most powerful due to its speed, sensitivity, and quantitative nature. With current instrumentation, 20-40 proteins can be measured per cell, at rates of thousands of cells per second. However, a major limitation of the platform is the number of antibody reagents that have been developed and validated for flow cytometry. Currently, less than 2% of intracellular protein targets can be measured by flow cytometry, while other biochemical techniques such as Western blotting are able to measure over 25%. This limitation in flow cytometry is largely due to the fact that the cells need to remain intact for analysis. This precludes sample preparation methods that would fully denature the proteins and expose the epitopes necessary for detection by a wider range of antibodies. Conversely, in Western blotting, the proteins are denatured and spread out on a membrane surface, making them accessible to antibodies which have been generated against linearized epitopes. Here we propose a technology platform that transfers the proteome of a single cell into a hydrogel bead. This enables the proteins to be denatured fully with harsh denaturation treatments, while also spreading them out in space to allow for easier access by antibody reagents, as the beads are larger than the original cells. The system is designed to preserve the critical advantages of single cell analysis by flow cytometry, such as the ability to stain for surface markers in complex immune cell populations, speed of acquisition, and detection sensitivity. The beads are also uniform in size, stable in solution, and optically clear, creating an ideal substrate for highly sensitive detection. By emulating the environment typically encountered in Western blots, we anticipate that the single cell bead platform will enable the use of hundreds to thousands of additional antibody reagents. This vast expansion in available reagents will enable next generation single cell analysis, the direct analysis of proteins instead of downstream or upstream surrogate markers, an increase in the number of signaling pathways that can be monitored, and ultimately deeper insight into disease states and drug activity.