Investigating Physicochemical Principles for Sample Preparation and Detection From Single-cell Immunoblots

Proteins are complex molecules that carry out diverse actions in cells for proper structure, function, and regulation in any organism. Proteins are encoded by genes, but can undergo modifications not encoded by DNA, that result in differences in function, even amongst proteins of the same type. These modifications beget protein isoforms, or variants of the same protein, that can have different biological functions and alter the behavior of an individual cell. Differences at the single-cell level can result in subpopulations of cells of heterogeneous behavior that can influence stem cell differentiation, disease development, and the efficacy of drug treatments. In order to understand and predict these phenomena, it is important to detect protein isoforms at the single-cell level. However, specific detection of the modifications that distinguish protein isoforms with single-cell resolution sensitivity remains challenging. To address the challenge of protein isoform detection, single-cell electrophoretic cytometry (scEPC) methods have been developed. scEPC leverages microscale transport phenomena and timescales to perform separations on individual cells to distinguish protein isoforms prior to detection via conventional immunoassay. Here, we advance our understanding of scEPC to guide assay design./nFirst, we characterize separation and detection performance with different gel pore size to interrogate large ranges of protein molecular masses that are relevant to protein signaling pathways. Furthermore, through our understanding of mathematical models established for bulk, (non-single cell) separations, we elucidate and identify anomalous protein electromigration behavior for large molecular mass proteins. The anomalous migration indicates further optimization of the cell lysis and protein solubilization sample preparation steps of our assay is potentially required for larger molecular mass targets./nNext, we aim to reduce fluorescence background for in-gel immunoassays that results from using benzophenone as a photoactivatable molecule for immobilizing protein in the gel matrix for subsequent fluorescence detection. We hypothesize that the benzophenone chemical structure, which contains two aromatic rings, contributes to increased fluorescence background signal due to autofluorescence and increased hydrophobic interactions that non-specifically interact with fluorescent immunoprobes to retain the immunoprobes in the gel matrix. We investigate an alternative photoactivatable molecule, diazirine, a three-membered ring comprised of two nitrogen atoms and one carbon atom, to reduce background fluorescence signal and enable detection lower abundance isoforms. Due to the chemical structure diazirine, we hypothesize that diazirine has lower autofluorescence compared to benzophenone. Furthermore, diazirine is a more hydrophilic molecule, which we hypothesize will reduce non-specific interaction with immunoprobes compared to benzophenone. Thus, we determine how to appropriately evaluate and compare diazirine and benzophenone, with regard to metrics important for in-gel immunoassay fluorescence background and signal./nLastly, we begin preliminary investigations to measure subcellular estrogen receptor-α (ERα) isoform expression from the cell membrane, cytoplasm, and nucleus using two approaches. To detect membrane-localized isoforms, we utilize a ligand that binds to ERα and is conjugated to a bulky molecule to pull down and isolate membrane-localized protein. To differentiate cytoplasmic and nuclear localized isoforms, we investigate efficacy of employing differential detergent fractionation chemistry and bi-directional electrophoretic separations. We anticipate that the finding presented here will advance assay capability to detect a wide range of protein isoform targets.

Advisor: Herr, Amy E