Post-translational modifications (PTMs) of receptor tyrosine kinases (RTKs) at the plasma membrane (PM) determine the signal transduction efficacy alone and in combination. However, current approaches to identify PTMs provide ensemble results, inherently overlooking combinatorial PTMs in a single polypeptide molecule. Here, we describe a single-molecule blotting (SiMBlot) assay that combines biotinylation of cell surface receptors with single-molecule fluorescence microscopy. This method enables quantitative measurement of the phosphorylation status of individual membrane receptor molecules and colocalization analysis of multiple immunofluorescence signals to directly visualize pairwise site-specific phosphorylation patterns at the single-molecule level. Strikingly, application of SiMBlot to study ligand-dependent epidermal growth factor receptor (EGFR) phosphorylation, which is widely thought to be multi-phosphorylated, reveals that EGFR on cell membranes is hardly multi-phosphorylated, unlike in vitro autophosphorylated EGFR. Therefore, we expect SiMBlot to aid understanding of vast combinatorial PTM patterns, which are concealed in ensemble methods, and to broaden knowledge of RTK signaling.

We present that modulation of fluorescence emission by linearly polarized excitation light can allow us to resolve spatially two fluorescent molecules within a diffraction limit and to determine simultaneously their precise dipole directions. Using polarization-dependent photoswitching, we imaged the 2D geometry of the DNA Holliday junction in a 10-nm length scale by measuring both the distance and the in-plane dipole angle between Cy3 emitters stacked onto the ends of two adjacent branches of the Holliday junction. The proposed polarization-modulated imaging technique provides a simple and nonstochastic imaging process to visualize the nanostructure, including directional information, of biomolecules beyond the diffraction limit.