Upconverting Nanoparticle-Based Photoactive Probes for Highly Efficient Labeling and Isolation of Target Proteins

Photoaffinity labeling (PAL) has blossomed into a powerfulandversatile tool for capture and identification of biomolecular targets.However, low labeling efficiency for specific targets such as lectins,the tedious process for protein purification, inevitable cellularphotodamage, and less tissue penetration of UV light are significantchallenges. Herein, we reported a near-infrared (NIR) light-drivenphotoaffinity labeling approach using upconverting nanoparticle (UCNP)-basedphotoactive probes, which were constructed by assembling photoactivegroups and ligands onto NaYF4:Yb,Tm nanoparticles. Thenovel probes were easily prepared and functionalized, and the labeledproteins can be isolated and purified through simple centrifugationand washing. The advantages of this approach were demonstrated bylabeling and isolation of peanut agglutinin (PNA), asialoglycoproteinreceptor (ASGPR), and human carbonic anhydrase II (hCAII) from mixedproteins or cell lysates with good selectivity and efficiency, especiallyfor PNA and ASGPR, two lectins that showed low binding affinity totheir ligands. More importantly, successful labeling of PNA throughpork tissues and ASGPR in mice strongly proved the good tissue penetratingcapacity of NIR light and the application potential of UCNP-basedphotoactive probes for protein labeling in vivo.Biosafety of this approach was experimentally validated by enzyme,cell, and animal work, and we demonstrated that NIR light caused minimalphotodamage to enzyme activity compared to UV light, and the UCNP-basedphotoactive probe presents good biosafety both in vitro andin vivo. We believe that this novel PAL approach will providea promising tool for study of ligand-protein interactions andidentification of biomolecular targets.

Automated summary

A near-infrared light-driven photoaffinity labeling approach using upconverting nanoparticle-based photoactive probes is developed for efficient and selective protein labeling. The labeled proteins can be easily isolated and purified, and the method demonstrates good tissue penetrating capacity for in vivo protein labeling.