Construction of a Mass-Tagged Oligo Probe Set for Revealing Protein Ratiometric Relationship Associated with EGFR-HER2 Heterodimerization in Living Cells.

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Protein dimerization, as the most common form of protein-protein interaction, can manifest more significant roles in cellular signaling than individual monomers. For example, excessive formation of EGFR-HER2 dimer has been implicated in cancer development and therapeutic resistance in addition to the overexpression of EGFR and HER2 proteins. Thus, quantitative evaluation of these heterodimers in living cells and revelation of their ratiometric relationship with protein monomers in dimerization may provide insights into clinical cancer management. To achieve this goal, the prerequisite is protein heterodimer quantification. Given the current lack of quantitative methods, we constructed a mass-tagged oligo nanoprobe set for quantification of EGFR-HER2 dimer in living cells. The mass-tagged oligo nanoprobe set contained two targeting probes (nucleic acid aptamers), a connector probe, a hairpin probe, and a photocleavable mass-tagged probe. Two distinct aptamers can recognize target protein monomers and initiate the subsequent hybridization cascade involving binding to the connector probe, formation of an initiator strand, opening of a hairpin probe, and ensuing hybridization with a photocleavable mass-tagged probe. Ultimately, the mass tag was released under ultraviolet light and then subjected to mass spectrometric analysis. In this way, the information regarding the interaction between two protein monomers was successfully converted to the quantitative signal of the mass tag. Using the assay, the expression level of EGFR-HER2 dimer and its relationship with individual protein monomers were determined in four breast cancer cell lines. We are among the first to obtain the absolute level of protein heterodimer, and this quantitative information may be vital in understanding the molecular basis of cancer.