Single-molecule blotting to detect site-specific receptor phosphorylations

Single-molecule blotting to detect site-specific receptor phosphorylations


Receptor tyrosine kinases (RTKs) play important roles in diverse biological functions, including cell-to-cell communication, proliferation and signal propagation, by their inherent kinase activity. Most biological processes, including RTK signaling, are coordinated by protein regulation such as post-translational modifications (PTMs), many of which provide binding sites for specific protein–protein interactions and signaling complex formation. Understanding how signaling receptor molecules are dynamically modified has helped to elucidate their roles in cellular function and regulation.

To determine the characteristics of specific protein pools, conventional methods, such as western blotting and mass spectrometry (MS), are widely used. Tremendous technological advances in biochemical and proteomic approaches achieved the identifications of more than 400 discrete types of modifications and 90,000 individual PTMs.

However, existing ensemble methods are virtually inapplicable to detect the combination of PTM sites on a single polypeptide molecule, the so-called ‘PTM code, which may confer different properties and functions. They suffer from inherent problems including ensemble averaging, loss of intact protein information, stochastic site assignment of combinatorial modification pattern and laborious and high-cost assay. Therefore, analysis of site-specific PTM patterns within individual protein molecules is still unexplored and remains challenging.

Recently, the emerging development of single-molecule techniques enables the observation and characterization of individual molecules for exquisite qualitative and quantitative analysis, avoiding ensemble error. Single-molecule techniques are well suited for characterizing multiple PTMs dispersed along the entire protein sequence but no feasible method exists.

One promising approach is single-molecule imaging combined with immunofluorescence labeling, which may yield quantitative measurement of PTM status at the single-molecule level. Methods based on super-resolution imaging in intact cells cannot control the intrinsic density of interesting protein, preventing the discrimination of individual modified proteins by high molecular density on the PM. However, several limitations have hampered the application of single-molecule isolation techniques to the study of combinatorial PTMs.

Researchers in the journal Nature Communications describe a simple, ultra-rapid and low-cost single-molecule assay with an antibody-free immobilization to investigate combinatorial PTMs of RTKs, named as ‘Single-MoleculeBlotting’ (SiMBlot). SiMBlot can directly immobilize biotinylated cell surface proteins on the single-molecule surface and enables the pairwise immunofluorescence labeling to detect multi-site PTMs of a single polypeptide molecule.

To demonstrate the unique power of this approach, they applied SiMBlot to reveal the pairwise site-specific phosphorylation patterns of individual EGFR molecules, which are extracted from the cell surface membrane in response to the EGF stimulus or sampled from an in vitro autophosphorylation assay.

These results call into question ligand-dependent multi-phosphorylation of EGFR, which is popularly believed to occur, and provide an insight into the molecular mechanism underlying EGFR activation

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