Nanoelectrospray ionization emitters with submicron tip diameters have significant advantages for use in native mass spectrometry, including the ability to produce resolved charge-state distributions for proteins and macromolecular complexes from standard biochemical buffers that contain high concentrations of nonvolatile salts and to prevent nonspecific aggregation that can occur during droplet evaporation. We report on various factors affecting the tip morphology and provide suggestions for producing and using emitters with submicron tips. Effects of pulling parameters for a Sutter Instrument P-87 tip puller on the resulting tip diameter and morphology are shown. The “Pull” parameter has the largest effect on tip diameter, followed by “Velocity”, “Pressure”, and “Heat”, whereas the “Time” parameter has minimal effect beyond a lower threshold. High “Pull” values generate emitters with multiple tapers, whereas high “Velocity” values generate a tip with only a single tapered region. A protocol for producing reproducible emitters in the submicron size range, as well as guidelines and tips for using these emitters with standard biochemical buffers that contain high concentrations of nonvolatile salts, is presented with the aim of expanding their use within the native mass spectrometry community.

The sizes and shapes of nanoparticles play a critical role in their chemical and material properties. Common sizing methods based on light scattering or mobility lack individual particle specificity, and microscopy-based methods often require cumbersome sample preparation and image analysis. A promising alternative method for the rapid and accurate characterization of nanoparticle size is charge detection mass spectrometry (CDMS), an emerging technique that measures the masses of individual ions. A recently constructed CDMS instrument designed specifically for high acquisition speed, efficiency, and accuracy is described. This instrument does not rely on an ion energy filter or estimates of ion energy that have been previously required for mass determination, but instead uses direct, in situ measurements. A standardized sample of ∼100 nm diameter polystyrene nanoparticles and ∼50 nm polystyrene nanoparticles with aminefunctionalized surfaces are characterized using CDMS and transmission electron microscopy (TEM). Individual nanoparticle masses measured by CDMS are transformed to diameters, and these size distributions are in close agreement with distributions measured by TEM. CDMS analysis also reveals dimerization of ∼100 nm nanoparticles in solution that cannot be determined by TEM due to the tendency of nanoparticles to agglomerate when dried onto a surface. Comparing the acquisition and analysis times of CDMS and TEM shows particle sizing rates up to ∼80× faster are possible using CDMS, even when samples ∼50× more dilute were used. The combination of both high-accuracy individual nanoparticle measurements and fast acquisition rates by CDMS represents an important advance in nanoparticle analysis capabilities.

Spraying solutions of serine under a wide variety of conditions results in unusually abundant gaseous octamer clusters that exhibit significant homochiral specificity, but the extent to which these clusters exist in solution or are formed by clustering during droplet evaporation has been debated. Electrospray ionization emitters with tip sizes between 210 nm and 9.2 μm were used to constrain the number of serine molecules that droplets initially contain. Protonated octamer was observed for all tip sizes with 10 mM serine solution, but the abundance decreases from 10% of the serine population at the largest tip size to ∼5.6% for the two smallest tip sizes. At 100 μM, the population abundance of the protonated serine octamer decreases from 1% to 0.6% from the largest to the smallest tip size, respectively. At 100 μM, fewer than 10% of the initial droplets should contain even a single analyte molecule with 210 nm emitter tips. These results indicate that the majority of protonated octamer observed in mass spectra under previous conditions is formed by clustering inside the electrospray droplet, but ≤5.6% and ∼0.6% of serine exists as an octamer complex in 10 mM and 100 μM solutions, respectively. These results show that aggregation occurs in large droplets, but this aggregation can be eliminated using emitters with sufficiently small tips. Use of these emitters with small tips is advantageous for clearly distinguishing between species that exist in solution and species formed by clustering inside droplets as solvent evaporation occurs.

Temperature-controlled nanoelectrospray ionization has been used to measure heat-induced conformational changes of biomolecules by mass spectrometry, but long thermal equilibration times associated with heating or cooling an entire emitter limit how fast these data can be acquired. Here, the tip of a borosilicate electrospray emitter is heated using 10.6 μm light from an unfocused CO2 laser. At 1.2 W, the solution inside the emitter tip can be heated from room temperature to a steady-state temperature of 78.2 ± 2.5 °C in less than 0.5 s and cools from 82.6 ± 0.6 °C back to room temperature within 4 s. The time required to establish a steady-state temperature is more than 100-fold faster than that required for a resistively heated emitter due to the low thermal mass. Protein unfolding curves measured as a function of laser power can be acquired in ∼40 s compared to a resistively heated apparatus that required ∼21 min to acquire similar data. Laser power is calibrated to temperature by comparisons of the average charge state of the protein cytochrome c measured with laser heating and with resistive heating. This laser heating method is applied to a three-component protein mixture to demonstrate the ability to rapidly acquire melting temperatures of proteins in mixtures. The ability to rapidly assess the thermal stabilities of multiple proteins simultaneously shows significant promise for coupling temperature-controlled electrospray ionization (ESI) to separation techniques, providing a high-throughput method for determining the effects of solution composition, drug binding, or sequence mutations on protein thermal stability.

The ability of electrospray emitters with submicron tip diameters to significantly reduce and even eliminate aggregation of analyte molecules that can occur inside evaporating droplets was recently demonstrated to show...