Combinatorial
post-translational modifications (PTMs), which can
serve as dynamic “molecular barcodes”, have been proposed
to regulate distinct protein functions. However, studies of combinatorial
PTMs on single protein molecules have been hindered by a lack of suitable
analytical methods. Here, we describe erasable single-molecule blotting
(eSiMBlot) for combinatorial PTM profiling. This assay is performed
in a highly multiplexed manner and leverages the benefits of covalent
protein immobilization, cyclic probing with different antibodies,
and single molecule fluorescence imaging. Especially, facile and efficient
covalent immobilization on a surface using Cu-free click chemistry
permits multiple rounds (>10) of antibody erasing/reprobing without
loss of antigenicity. Moreover, cumulative detection of coregistered
multiple data sets for immobilized single-epitope molecules, such
as HA peptide, can be used to increase the antibody detection rate.
Finally, eSiMBlot enables direct visualization and quantitative profiling
of combinatorial PTM codes at the single-molecule level, as we demonstrate
by revealing the novel phospho-codes of ligand-induced epidermal growth
factor receptor. Thus, eSiMBlot provides an unprecedentedly simple,
rapid, and versatile platform for analyzing the vast number of combinatorial
PTMs in biological pathways.