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

Abstract

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.

Steric Hindrance On–Off Mass-Tagged Probe Set Enables Detection of Protein Homodimer in Living Cells

“…Among a wide variety of mass tags, well-designed peptides have gained more interest because they have both unique sequence to avoid overlapping with endogenous peptides and sufficient mass spectrometric signal for quantitative analysis . The peptide AVLGDPFR was selected as the mass tag . Finally, another photocleavable linker, o -nitrobenzyl derivative PL2 , was used to ligate the mass tag and HB5 aptamer to generate the detection probe HB5- PL2 -mass tag.…”

“…35 The peptide AVLGDPFR was selected as the mass tag. 36 Finally, another photocleavable linker, onitrobenzyl derivative PL2, 37 was used to ligate the mass tag and HB5 aptamer to generate the detection probe HB5-PL2mass tag. S1).…”

Mass tag-encoded nanointerfaces for multiplexed mass spectrometric analysis and imaging of biomolecules

“…In some of the MT-encoded nanointerfaces, recognition modules are necessary for identifying or responding to the target molecules to establish the correspondence between MTs and targets. So far, oligonucleotides, 28,[43][44][45] peptides, 21,46 antibodies, 17,42,47 nucleic acid aptamers, 26,48,49 and lectins 50 have been employed as biometric components conjugated with MTs through a linkage, aiming at specific mass tagging of multiple target biomolecules.…”

Amplified and Specific Staining of Protein Dimerization on Cell Membrane Catalyzed by Responsively Installed DNA Nanomachines for Cancer Diagnosis

“…Multiple DNA logic gates can be connected into complex signaling cascades to develop synthetic circuits based on nucleic acid hybridization, which have been applied to the analyzation of protein-protein interactions on cell surface. [18][19][20][21][22] In order to transform the recognition information into visual signal, many DNA computation devices integrated with G-quadruplex-Hemin DNAzyme were developed. [23] G-quadruplexes (G4s) consisting of stacks of G-tetrads which are cycle planar arrangement of four Hoogsteen hydrogen-bonded guanine residues, could bind to Hemin and effectively catalyze the H 2 O 2 -mediated oxidation of several substrates accompanied by a color change.…”