Nanobead-based single-molecule pulldown for single cells

Investigation of cell-to-cell variability holds critical physiological and clinical implications. Thus, numerous new techniques have been developed for studying cell-to-cell variability, and these single-cell techniques can also be used to investigate rare cells. Moreover, for studying protein-protein interactions (PPIs) in single cells, several techniques have been developed based on the principle of the single-molecule pulldown (SiMPull) assay. However, the applicability of these single-cell SiMPull (sc-SiMPull) techniques is limited because of their high technical barrier and special requirements for target cells and molecules. Here, we report a highly innovative nanobead-based approach for sc-SiMPull that is based on our recently developed microbead-based, improved version of SiMPull for cell populations. In our sc-SiMPull method, single cells are captured in microwells and lysed in situ, after which commercially available, pre-surface-functionalized magnetic nanobeads are placed in the microwells to specifically capture proteins of interest together with their binding partners from cell extracts; subsequently, the PPIs are examined under a microscope at the single-molecule level. Relative to previously published methods, nanobead-based sc-SiMPull is considerably faster, easier to use, more reproducible, and more versatile for distinct cell types and protein molecules, and yet provides similar sensitivity and signal-to-background ratio. These crucial features should enable universal application of our method to the study of PPIs in single cells. Statement of SignificanceHeterogeneity between single cells holds critical physiological and clinical implications. Characterization of protein-protein interactions (PPIs) and identification of the interacting partners of a specific protein are critical for elucidating the function and regulation of the protein. However, the applicability of the currently available techniques for studying PPIs in single cells is limited by their high technical barrier and special requirements for cell/proteins types. Our single-cell single-molecule pulldown (sc-SiMPull) assay in this study is not only substantially simpler and faster than existing sc-SiMPull methods, but also considerably more widely applicable--to all cell types and to both soluble and transmembrane proteins. These two crucial features should enable universal application of our method to the study of PPIs in single cells.