Improved Mass Analysis of Proteins and Protein Complexes Present in Common Biological Buffers and Physiological...

Native electrospray ionization-mass spectrometry (ESI-MS) is inherently susceptible to nonvolatile salts in solution, due to ion formation resulting from the so-called "charged-residue mechanism" (CRM). The presence of nonvolatile salts leads to peak broadening and shifting to higher mass due to salt condensation onto the analyte, and in the worst-case scenario, can totally suppress the observation of the analyte ions of interest. However, salts are known to play roles in protein conformation and dynamics. Previously, we showed 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 biological buffers and nonvolatile salts at physiologically relevant concentrations. However, the signal-to-noise (S/N) ratios of the bio-ions of interest were significantly lower compared with the bio-ions' signals in the absence of biological buffers. Here, implementing theta emitters for sample introduction, we show how the delivery of anions with relatively low proton affinities during the ESI droplet formation can significantly reduce ionization suppression through mitigation of chemical noise. Importantly, this strategy increases S/N ratios, method reproducibility, and robustness compared to our previous work. These advantages are important for the mass analysis of protein complexes extracted from biological tissues, where the amount of starting material is limited.