Kim Kyung Lock, Kim Daehyung, Lee Seongsil, Kim Su-Jeong, Noh Jung Eun, Kim Joung-Hun, Chae Young Chan, Lee Jong-Bong, Ryu Sung Ho
Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
Nat Commun. 2016 Mar 24;7:11107. doi: 10.1038/ncomms11107.
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.
质膜（PM）上受体酪氨酸激酶（RTK）的翻译后修饰（PTM）单独或共同决定信号转导效率。然而，目前鉴定PTM的方法提供的是总体结果，本质上忽略了单个多肽分子中的组合PTM。在这里，我们描述了一种单分子印迹（SiMBlot）分析方法，该方法将细胞表面受体的生物素化与单分子荧光显微镜相结合。这种方法能够定量测量单个膜受体分子的磷酸化状态，并对多个免疫荧光信号进行共定位分析，以在单分子水平直接可视化成对的位点特异性磷酸化模式。引人注目的是，将SiMBlot应用于研究配体依赖性表皮生长因子受体（EGFR）磷酸化（普遍认为其为多磷酸化）时发现，与体外自磷酸化的EGFR不同，细胞膜上的EGFR几乎没有多磷酸化。因此，我们期望SiMBlot有助于理解隐藏在总体方法中的大量组合PTM模式，并拓宽对RTK信号传导的认识。
