In Situ Simultaneous Profiling Of Phosphorylation And Ubiquitination

Phosphorylation and ubiquitination are the most prevalent among post-translational modifications(PTMs).Both reversible covalent modifications can be combined sequentially or simultaneously as binding partners to alter the function and localization of proteins.More significantly,the regulation of protein ubiquitination depends on protein phosphorylation.In order to precisely regulate intracellular protein functions,it is critical to monitor phosphorylation and ubiquitination simultaneously with the complement of each other.Analytical techniques used for the analysis of protein phosphorylation or/and ubiquitination usually resort to instrumental methods of mass spectrometry and western blot analysis using antibodies.But,these studies could realize detection of one single type of protein owing to the limitations of highly specific enrichment methods.Since fluorescence probes are the desirable analytical approach in biotechnology,several commercial probes have being developed for specific fluorescent detection of phosphorylation alone.However,these probes perform well in vitro and can only focus on one modification,and no methods have ever been developed to in situ monitor phosphorylation and ubiquitination simultaneously using specific probes.Lanthanide-doped UCNPs utilize sequential absorption of multiple photons,which are generally located in the near-infrared(NIR)region,to produce short wavelength luminescence emission.Owing to their unique properties,UCNPs provide excellent NIR-activated multi-color emissions.In this work,we prepared a dual-emission UCNP for simultaneous profiling of phosphorylation and ubiquitination of target protein on the cytomembrane using HER2 protein,a transmembrane tyrosine kinase receptor in the human EGFR family that regulates cell differentiation and proliferation,and is overexpressed in many cancers,as the model.HER2 could be recognized by aptamer-functionalized UCNP on living cell surface due to the selective binding affinity of the 86-base-aptamer toward the HER2 protein.The two types of PTMs(phosphorylation and ubiquitination)on the HER2 protein were monitored by synthetic cyanine 3(Cy3)linked dizinc(II)fluorescent molecular probes and cyanine 5.5(Cy5.5)linked Ub antibodies,respectively.Upon a single excitation at 980 nm,two energy emission bands of UCNP were regarded as two independent energy donors.The fluorescence of acceptors,cyanine 3(Cy3)and Cy5.5,linked to HER2 could be lit up via luminescence resonance energy transfer process for synergistic imaging of phosphorylation and ubiquitination.Moreover,the relative quantification of phosphorylation and ubiquitination could be used to distinctly visualize their interactions along with HER2 degradation on various cancer cell types,thus providing a new insight into the protein degradation-related apoptosis pathway.