Evaluation of Site-Diversified, Fully Functionalized Diazirine Probes for Chemical Proteomic Applications

Photoaffinity probes combined with the chemical proteomic platform have emerged as versatile tools for ligand and target discovery. However, photoaffinity probes with retained activity cannot always label the known target, indicating that it is challenging to profile a ligand’s targets based on its photoaffinity probe modified at a single site. Herein, we construct a series of site-diversified probes (P1-P6) of 4-anilinoquinazoline, a scaffold shared by several marketed EGFR-targeted drugs, via attaching a “fully functionalized” diazirine tag to six different sites, respectively. Chemical proteomic analysis revealed that these probes show different proteome-wide profiles and distinct competition patterns by erlotinib. Remarkably, low activity P4 towards EGFR inhibition has better EGFR labelling efficiency than the higher one, P5, which highlights the dominance of labelling accessibility of diazirine over probe affinity. In addition, the integrated analysis of protein targets of site-diversified probes can also help distinguish false positive targets. We anticipate that site-diversification of the probes of a given scaffold is an indispensable strategy to truly harness the power of photoaffinity-based chemoproteomics in drug discovery.

Evaluation of Site-Diversified, Fully Functionalized Diazirine Probes for Chemical Proteomic Applications

Photoaffinity probes combined with the chemical proteomic platform have emerged as versatile tools for ligand and target discovery. However, photoaffinity probes with retained activity cannot always label the known target, indicating that it is challenging to profile a ligand’s targets based on its photoaffinity probe modified at a single site. Herein, we construct a series of site-diversified probes (P1-P6) of 4-anilinoquinazoline, a scaffold shared by several marketed EGFR-targeted drugs, via attaching a “fully functionalized” diazirine tag to six different sites, respectively. Chemical proteomic analysis revealed that these probes show different proteome-wide profiles and distinct competition patterns by erlotinib. Remarkably, low activity P4 towards EGFR inhibition has better EGFR labelling efficiency than the higher one, P5, which highlights the dominance of labelling accessibility of diazirine over probe affinity. In addition, the integrated analysis of protein targets of site-diversified probes can also help distinguish false positive targets. We anticipate that site-diversification of the probes of a given scaffold is an indispensable strategy to truly harness the power of photoaffinity-based chemoproteomics in drug discovery.

Direct digital sensing of proteins in solution through single-molecule optofluidics

Highly sensitive detection of proteins is of central importance to biomolecular analysis and diagnostics. Conventional protein sensing assays, such as ELISAs, remain reliant on surface-immobilization of target molecules and multi-step washing protocols for the removal of unbound affinity reagents. These features constrain parameter space in assay design, resulting in fundamental limitations due to the underlying thermodynamics and kinetics of the immunoprobe–analyte interaction. Here, we present a new experimental paradigm for the quantitation of protein analytes through the implementation of an immunosensor assay that operates fully in solution and realizes rapid removal of excess probe prior to detection without the need of washing steps. Our single-step optofluidic approach, termed digital immunosensor assay (DigitISA), is based on microfluidic electrophoretic separation combined with single-molecule laser-induced fluorescence microscopy and enables calibration-free in-solution protein detection and quantification within seconds. Crucially, the solution-based nature of our assay and the resultant possibility to use arbitrarily high probe concentrations combined with its fast operation timescale enables quantitative binding of analyte molecules regardless of the capture probe affinity, opening up the possibility to use relatively weak-binding affinity reagents such as aptamers. We establish and validate the DigitISA platform by probing a biomolecular biotin–streptavidin binding complex and demonstrate its applicability to biomedical analysis by quantifying IgE–aptamer binding. We further use DigitISA to detect the presence of α-synuclein fibrils, a biomarker for Parkinson’s disease, using a low-affinity aptamer at high probe concentration. Taken together, DigitISA presents a fundamentally new route to surface-free specificity, increased sensitivity, and reduced complexity in state-of-the-art protein detection and biomedical analysis.