Integrated electrophoretic cytometry separations and immunoassays for proteins and their complexes

Protein complexes, such as filamentous actin (F-actin) complexes, regulate key cell processes such as cell motility and division. Disruption of F-actin result in highly motile and invasive cancer cells. Cancer therapeutics have thus aimed to maintain F-actin, but cell-to-cell variation in F-actin levels in response to such therapeutics necessitate single-cell measurements of dynamic actin protein complexes, including the binding actin binding proteins that determine actin polymerization state. Protein complex levels cannot be inferred from an immunoassay, as most lack selective antibodies. Size-based separations of such protein species provide selectivity when coupled with an immunoassay for protein detection and quantitation. While this selectivity has been demonstrated at the single-cell level by the introduction of electrophoretic (EP) cytometry in our lab, we sought to establish a single-cell electrophoretic assay for protein complex identification and quantitation. In order to understand the regulation of actin polymerization and depolymerization in heterogeneous cells requires four key separation assay features: i) quantifiable technical variation to discern biological variation in the cell population ii) sufficient analytical sensitivity to detect F-actin bound actin binding proteins, iii) high-selectivity separations to detect actin and its binding proteins, and iv) sample preparation with assay stage-optimized buffers to isolate dynamic complexes without disrupting the complexes. We will share our studies to elucidate chemical and physical underpinnings of each of these needed features. First, we will describe algorithm development and applications to establish a technical variation threshold and protein sizing standards for electrophoretic (EP) cytometry to distinguish biological variation of protein expression and size in single cells. Next, we will discuss the impact of in-gel immunoassay performance and open microfluidic device design on analytical sensitivity. Given fundamental tradeoffs between in-gel immunoassay sensitivity and separation performance, we consider alternative sieving matrices tuned to separate proteins in specific molecular weight ranges. We then describe unique impacts of Joule heating on separation performance in open microfluidic electrophoresis. Joule heating is mitigated with a buffer exchange approach that reduces variation in separation performance and introduces assay stage-optimized buffers without further protein loss. Finally, we will discuss the design of EP cytometry to fractionate actin protein complexes from single cells with assay stage-optimized buffers. The microscale device achieves rapid, arrayed on-chip sample preparation and EP fractionation without perturbing complexes. We demonstrated F-actin separations from monomeric actin, and the measurement of F-actin binding proteins that regulate actin polymerization. We anticipate the single-cell protein complex measurements described here will be broadly applicable to protein complexes that drive human health