Electrospray ionization (ESI) mass spectrometry (MS) is a crucial method for rapidly determining the interactions between small molecules and proteins with ultrahigh sensitivity. However, nonvolatile molecules and salts that are often necessary to stabilize the native structures of protein–ligand complexes can readily adduct to protein ions, broaden spectral peaks, and lower signal-to-noise ratios in native MS. ESI emitters with narrow tip diameters (∼250 nm) were used to significantly reduce the extent of adduction of salt and nonvolatile molecules to protein complexes to more accurately measure ligand–protein binding constants than by use of conventional larger-bore emitters under these conditions. As a result of decreased salt adduction, peaks corresponding to protein–ligand complexes that differ in relative molecular weight by as low as 0.06% can be readily resolved. For low-molecular-weight anion ligands formed from sodium salts, anion-bound and unbound protein ions that differ in relative mass by 0.2% were completely baseline resolved using nanoscale emitters, which was not possible under these conditions using conventional emitters. Owing to the improved spectral resolution obtained using narrow-bore emitters and an analytically derived equation, K d values were simultaneously obtained for at least six ligands to a single druggable protein target from one spectrum for the first time. This research suggests that ligand–protein binding constants can be directly and accurately measured from solutions with high concentrations of nonvolatile buffers and salts by native MS.

Netropsin is one of the first ligands to be discovered that selectively binds to the minor groove of DNA and is actively used as a scaffold for developing potential anticancer and antibiotic agents. The mechanism by which netropsin binds to hairpin DNA remains controversial with two competing mechanisms having been proposed. In one mechanism, netropsin binding induces a hairpin-to-duplex DNA transition. Alternatively, netropsin binds in two thermodynamically different modes at a single duplexed AATT site. Here, results from native mass spectrometry (MS) with nanoscale ion emitters indicate that netropsin can simultaneously and sequentially bind to both hairpin and duplex DNA. Duplex DNA was not detected using conventional MS with larger emitters because nanoscale emitters significantly reduce the extent of salt adduction to ligand–DNA complex ions, including in the presence of relatively high concentrations of nonvolatile salts. Based on native MS and polyacrylamide gel electrophoresis results, the abundances of hairpin and duplex DNA are unaffected by the addition of netropsin. By native MS, the binding affinities for five ligand–DNA and DNA–DNA interactions can be rapidly obtained simultaneously. This research indicates a “simultaneous binding mechanism” for the interactions of netropsin with DNA.

Optically addressable spin defects in hexagonal boron nitride (hBN) have become a promising platform for quantum sensing. While sensitivity of these defects is limited by their interactions with the spin environment in hBN, inefficient microwave delivery can further reduce their sensitivity. Here, we design and fabricate a microwave double arc resonator for efficient transferring of the microwave field at 3.8 GHz. The spin transitions in the ground state of VB− are coupled to the frequency of the microwave cavity, which result in enhanced optically detected magnetic resonance (ODMR) contrast. In addition, the linewidth of the ODMR signal further reduces, achieving a magnetic field sensitivity as low as 42.4 μT/√Hz. Our robust and scalable device engineering is promising for future employment of spin defects in hBN for quantum sensing.