Bridging the Gap between Native Mass Spectrometry and Biochemistry: Small Droplets for Electrospray Ionization

Native mass spectrometry is widely used to study the structure, stoichiometry, stability, and interactions of biological macromolecules. In native mass spectrometry, intact biological molecules are directly transferred from solution into the gas phase. However, one disadvantage of native MS is the requirement for volatile buffers, such as ammonium acetate and ammonium bicarbonate, which differ from buffers that are commonly used in other biological methods, such as Tris buffer and phosphate buffered saline. Until very recently, most native mass spectrometry experiments have been done with volatile buffers until very recently. Nonvolatile salts, even at low tens of mM concentrations, can cause the suppression of analyte ions, an increased chemical baseline, and reduced resolution and accuracy of mass measurements. In some cases, no useful mass information is obtained due to the nonvolatile salts in the sample solution. Thus, the mismatched buffer choices between native mass spectrometry and conventional biochemical techniques raise questions about the results obtained with native MS. The development of submicron electrospray emitters enables acquisition of mass spectra with resolved charge state distributions, which can provide useful mass information, for proteins and protein complexes from solutions containing nonvolatile salts at physiologically relevant concentrations (≥ 150 mM NaCl). Submicron emitters have additional analytical advantages, including low sample consumption rate, providing results that are independent of instrument interface condition, and elimination of nonspecific dimer formation during electrospray ionization. With submicron emitters, the effects of ammonium acetate and traditional biological buffers on the structures, stabilities, and stoichiometries of proteins and protein complexes can be evaluated. The mass spectral results of the effect of salts on the protein and protein complexes are consistent with the discovery 130 years ago that salts can influence protein stability and solubility, which is known as the Hofmeister effect. This work demonstrates the use and advantages of submicron emitters. It is also the first study to provide mass spectral evidence that nonvolatile salts at a physiologically relevant concentration can influence protein structure, solubility, and stability, which has an important impact on the established native MS community that relies almost exclusively on volatile buffers. In addition to different emitter sizes that determine whether desalted protein ions can be formed from solutions containing high concentrations of nonvolatile salts during ESI, a different emitter design can provide unique advantages. This work will also discuss theta emitter as an alternative design of ESI emitters. Theta emitters with two separated barrels have the advantage that rapid mixing can be performed during electrospray ionization with controlled reaction time. The lifetime of droplets, which determines the reaction time, can range from 1 μs to several hundred microseconds. Different droplet lifetimes are achieved by varying the flow rate and the distance between the emitter and the instrument. Applications and phenomena related to theta emitters are shown in this work to demonstrate the advantages and limitations of theta emitters of different sizes, especially the submicron theta emitters. This work provides some insights on how to choose a suitable emitter size and design based on the need of the experiments, especially in the area of native MS