Abstract: The study of cellular heterogeneity of protein species, particularly of complexes that generally lack selective detection probes, has lagged behind nucleic acid single-cell analysis for several reasons. First, methods are insufficiently sensitive for detection of complexes from single-cell lysates. Second, techniques for single-cells such as FRET or proximity ligation assays are designed to analyze only two interacting proteins, while the vast majority of mammalian protein complexes are comprised of four or more subunits.
To address current shortcomings in single-cell analysis of protein complexes, we previously introduced the Single-cell protein Interaction Fractionation Through Electrophoresis and immunoassay Readout (SIFTER) assay. SIFTER separates protein complexes from monomeric proteins in hundreds of single-cell lysates simultaneously through size-exclusion electrophoresis in a microwell array. Separated monomeric proteins and dissociated protein complexes are immobilized in the array and detected with fluorescently labeled antibodies. While SIFTER enabled quantification of cytoskeletal protein complex heterogeneity, single cells were trypsinized for seeding in the microwell array, disrupting protein-complex interactions with the extracellular matrix. Furthermore, the fractionation step in SIFTER could not discern subunit composition of multicomponent protein complexes.
Here, we describe multiple advances in the analysis of protein complexes from single cells. First, we will share development of a novel single-cell encapsulation technique within extracellular matrix-mimicking hydrogel spheres that is compatible with SIFTER analysis. Second, we will describe the development of a multi-dimensional protein separation method to separate native protein complexes approximately by mass and charge before an orthogonal electrophoretic separation of the denatured protein complex subunits. Our high-throughput technologies to quantify expression of protein complexes from single cells have wide-ranging applicability to basic biomedical research and drug screening in conditions from cancer to neurodegeneration.