Characterization and Synthesis of Photoactive Hydrogels for Multiparameter Proteoform Measurements from Single Cells

Heterogeneity is a fundamental property of biological systems. The central dogma of biology posits that a gene encoded in DNA is transcribed into messenger RNA and translated to protein. Yet, a single gene does not necessarily map to a single protein. Protein isoforms (i.e., protein variants) produced during transcription or translation are subject to post-translational modifications, yielding a molecularly diverse collection of proteoforms. The molecular heterogeneity of proteoforms gives rise to the variable behavior of individual cells, and propagates across length scales to produce the readily observable traits that make each organism unique. Conversely, in the context of therapeutic interventions, elucidating why a treatment may elicit different responses in different individuals requires a fundamental understanding of biological heterogeneity at the cellular and molecular scales. Yet, the inverse relationship between specificity and analytical sensitivity makes discriminating between closely related proteoforms with single cell resolution a formidable challenge./nTo address the specificity challenge, our group pioneered electrophoretic separation assays from single cells, combining an immunoreactivity measurement with a simultaneous measurement of a second property (e.g., molecular mass, charge, cellular compartment). Such multiparameter measurements facilitate quantitation of non-specific antigen-antibody binding, and thus eliminate the need for proteoform-specific antibodies. Notably, proteoform detection in scEP assays occurs within a porous hydrogel matrix, and thus is subject to reaction-transport constraints. In this work, we apply fundamental principles of mass transport and reaction kinetics to in-gel enzyme-catalyzed and in-gel click chemistry reactions to expand the suite of measurable proteoform properties on the scEP platform./nFirst, given the prevalence of enzymes, an important class of protein chemical catalysts, as drug targets, we were interested in measuring enzymatic activity from single cells. Analogous to the non-specific antigen-antibody binding events that confound immunoassay signal, multiple distinct enzymes may act on the same substrate. As a result, we hypothesized that the upstream separation step in scEP assays can confer additional specificity to enzymatic activity measurements from single cells. To assess feasibility of measuring enzymatic activity from single cells, we studied the effect of immobilizing enzymes in UV-reactive hydrogel matrices on the intrinsic catalytic activity of the enzyme./nNext, we aimed to address the perennial tradeoff between specificity and analytical sensitivity in single cell proteoform measurements. The electrophoretic separation step in scEP assays spatially fractionates proteoforms according to a physical property (i.e., mass, charge). In the absence of proteoform-specific antibodies, the total signal is divided into discrete fractions. As a result, a simultaneous increase in analytical sensitivity is required to detect smaller protein quantities. To increase analytical sensitivity of the scEP platform, we evaluate an enzymatic signal amplification strategy for in-gel immunoassays./nLastly, to expand the measurement modalities compatible with scEP assays, we established a modular approach to fabricate chemically multifunctional hydrogels. UV-activated protein immobilization in hydrogels, which mitigates loss of proteins extracted from single cells, is central to the success of scEP assays. However, the current benzophenone-based immobilization approach is irreversible, limiting proteoform detection to single separation modalities (i.e., mass or charge) and in-gel detection with affinity probes. To support capabilities such as multi-dimensional separations and possible integration with off-gel proteoform detection, we developed a sequential click chemistry approach to confer reversible protein immobilization in hydrogels, among other chemical functions.

Advisor: Herr, Amy E