Multiplex single-cell analysis of intracellular protein signaling dynamics

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ABSTRACT Recent developments on single-cell analytical methods have provided a clarifying view on cellular heterogeneity and its associated mechanistic and therapeutic implications. Nevertheless, single-cell studies on intracellular protein signaling activities can be confounded by the dynamic nature of signaling events. In addition, there is a pressing need of developing non-genetic analytical methods for clinical samples. These requisites call for a single-cell approach that can interrogate intracellular protein signaling dynamics while being compatible with other downstream analytical methods. A recently developed prototype method has enabled dynamic probing of intracellular protein signaling activities at single-cell resolution, without genetic modifications. The technology was based on intracellularly delivered epitope-targeting peptide probes, a microwell-based single-cell chip and high-speed imaging techniques. However, many obstacles exist that challenge the generalizability of this approach and limit its biomedical application. In this proposal, the aim is to significantly advance this proof-of- concept technology. Five specific goals are included: develop an automated screening protocol for improved screening efficiencies, implement a unique bicyclic peptide library for higher biding affinities, employ cell permeabilizers as a more generalizable delivery method, achieve simultaneous analysis of multiple signaling proteins, and integrate this technology with other single-cell detection methods. This integrated multiplex pipeline will provide an enabling technology that promises a more in-depth understanding of the interplay among protein signaling activities, protein expression levels and metabolism in biological processes.