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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.
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Energy transfer chemiluminescence for ratiometric pH imaging
Chemiluminescence imaging offers a low background and high sensitivity approach to imaging analytes in living cells and animals. Intensity-based measurements have been developed, but require careful consideration of kinetics, probe localization, and fluctuations in quantum yield, all of which complicate quantification. Here, we report a ratiometric strategy for quantitative chemiluminescence imaging of pH. The strategy relies on an energy transfer cascade of chemiluminescence emission from a spiroadamantane 1,2-dioxetane to a ratiometric pH indicator via fluorescent dyes in Enhancer solutions. Monitoring the pH-dependent changes in chemiluminescence emission at multiple wavelengths enables ratiometric imaging and quantification of pH independent from variations due to kinetics and probe concentration.
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- Award ID(s):
- 1653474
- PAR ID:
- 10058544
- Date Published:
- Journal Name:
- Organic & Biomolecular Chemistry
- ISSN:
- 1477-0520
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/C8OB00972D
