Mass Spectrometry of Proteins and Protein Complexes Electrosprayed in the Presence of Common Biological Buffers Using Theta Emitters

Salt-protein interactions are essential in biology. However, the presence of nonvolatile salts at physiologically relevant concentrations (e.g., 150 mM NaCl) is detrimental to mass analysis by electrospray ionization mass spectrometry (ESI-MS). Nonvolatile salts tend to compromise ionization yields and, when analyte ions are observed, lead to peak broadening and shifts to higher mass. Ultimately, these phenomena yield lower signal-to-noise (S/N) ratios and, in the worst-case scenario, totally suppress the formation of the analyte ions of interest. For these reasons, the sample is generally desalted before mass analysis. Direct sample introduction into the mass spectrometer is widely used in "native" ESI-MS, where the preservation of protein interactions is a priority. Unfortunately, native ESI-MS is highly susceptible to nonvolatile salts in solution, and desalting steps might bring undesired consequences: sample loss, protein destabilization in solution, and the disruption or weakening of protein interactions. Here, we show how native ESI-MS implemented with theta emitters, glass emitters with a septum that divides the capillary into two channels, with inner diameters of ∼1.4 μm, allows for the identification of proteins and protein complexes in solutions containing nonvolatile salts at physiologically relevant concentrations. We posit that the differences arise from a statistical effect of incomplete mixing in a single Taylor cone; the small fraction of droplets that are relatively depleted in nonvolatile salts gives rise to the resolved analyte charge states. As a result, mass measurements of lysozyme (14 kDa), avidin (64 kDa), and beta-galactosidase (466 kDa) were enabled at physiologically relevant concentrations of nonvolatile salts.