title: Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting.
abstract: 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.
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Kyung Lock
authors.initials: KL
authors.lastname: Kim
authors.affiliation: Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Daehyung
authors.initials: D
authors.lastname: Kim
authors.affiliation: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Seongsil
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Su-Jeong
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Jung Eun
authors.initials: JE
authors.lastname: Noh
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Joung-Hun
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Young Chan
authors.initials: YC
authors.lastname: Chae
authors.affiliation: Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.|School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Jong-Bong
authors.initials: JB
authors.lastname: Lee
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.|School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Ryu
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors