Sample pH Can Drift during Native Mass Spectrometry Experiments: Results from Ratiometric Fluorescence Imaging

Abstract

The ability of nanoelectrospray ionization (nanoESI) to generate a continuous flow of charged droplets relies on the electrolytic nature of the process. This electrochemistry can lead to the accumulation of redox products in the sample solution. This consequence can have significant implications for native mass spectrometry (MS), which aims to probe the structures and interactions of biomolecules in solution. Here, ratiometric fluorescence imaging and a pH-sensitive, fluorescent probe are used to quantify changes in solution pH during nanoESI under conditions relevant to native MS. Results show that the extent and rate of change in sample pH depends on several experimental parameters. There is a strong correlation between the extent and rate of change in solution pH and the magnitude of both the nanoESI current and electrolyte concentration. Smaller changes in solution pH are observed during experiments when a negative potential is applied than for those when a positive potential is applied. Finally, we make specific recommendations for designing native MS experiments that control for these effects.

On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry

“…Inadvertent pH alterations during ESI can affect the outcome of experiments, e.g., by causing protein unfolding or the dissociation of complexes. ,− We will focus on the commonly used positive ion mode, where water oxidation 2 0.25em normalH 2 normalO → 4 0.25em normalH + + normalO 2 + 4 0.25em normale − is the main charge-balancing reaction, resulting in acidification of the solution in the emitter and ESI droplets . (We refer to protons in water as “H + ,” although it would be more appropriate to describe them as H 3 O + , or H 3 O + (H 2 O) n …”

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides

“…We anticipated that the spray solution would start near pH 6 or 10 and experience some level of buffering around pH 4.75 or 9.25 prior to ESI droplet desolvation. Increased buffer capacity is expected to maintain the droplet pH around the p K a of either acetate or ammonium during ESI, , which in turn is expected to enable more confident prediction of the pH surrounding the analyte during in-ESI HDX. This allows for characterization of how ESI affects labeling during in-ESI HDX.…”

Expanding Native Mass Spectrometry to the Masses

“…The sample solution pH can slowly change during electrospray, and can affect samples that are extremely sensitive to pH. 10,11 Finally for membrane proteins the detergent, membrane mimetic, pH, or cofactors are all important and can affect the oligomeric state observed. 12,13 There are multiple solution parameters, therefore, that may have to be optimized depending on the sample and the goal of the experiment.…”