Molecular Assemblies, Probes, and Proteomics in Flow Cytometry
Authors: Graves Steven W. , Nolan John P. 
Published: November 2020

The many proteins and nucleic acids encoded in the genome predominantly perform their functions as macromolecular assemblies. In fact, modern biomedical research often targets the interactions of individual molecules of these assemblies, usually by disrupting or enhancing specific contacts, to provide treatment for many different diseases. Therefore, efficient pharmaceutical design requires knowledge of how macromolecular assemblies are built and function. To achieve this goal, sensitive and quantitative tools are essential. This chapter will discuss the use of flow cytometry as a general platform for sensitive measurement and quantification of molecular assemblies. First, this chapter will introduce general methods for analysis of molecular interactions along with a comparison of flow cytometry with these methods. Second, an overview of current flow cytometry instrumentation, assay technologies, and applications in molecular assembly analysis will be given. Third, the implementation of the above approaches in molecular assembly will be discussed. Finally, potential future directions of flow cytometry in molecular assembly analysis will be explored. At present, the analysis of macromolecular assemblies is performed by a wide variety of techniques that are chosen for the target molecules under study (proteins, DNA, lipids, etc.), the type of measurement required (kinetic or equilibrium), and whether the assembly of interest needs to be studied in vivo or in vitro. This continuum of techniques can be divided into the heterogeneous assays, which require a separation step to resolve products from reactants, and homogeneous assays, which can measure interactions without a separation step. Heterogeneous assays, in general, use radioisotopes, which are not perturbing; offer excellent sensitivity; and provide accurate quantification. The products are quantified after a separation step such as gel filtration, gel electrophoresis, or centrifugation. Rapid quench methods can provide subsecond kinetic resolution; however, the added separation steps are tedious and make collection of kinetic time courses difficult, as each time point must be separated and measured individually. Furthermore, in the time it takes the separation to occur, the interaction of interest can dissociate, which is a problem specific to low-affinity assemblies. Nonetheless, by using rapid chemical quench techniques, reaction times as short as a few milliseconds can be observed. Homogenous assays can be separated into solution- or surface-based assays. Solutionbased assays measure an optical signal generated by the assembly to quantify an interaction. High component concentrations (micromolar) allow changes in intrinsic molecular properties, such as protein fluorescence or circular dichroism, to be used to study molecular assemblies. For greater sensitivity (nanomole component concentrations), resonance energy transfer or polarization assays using exogenous fluorescent labels can be used. In combination with stopped-flow spectroscopy methodologies, solution-based assays allow reactions to monitored in a continuous fashion with submillisecond dead times.