Related Experiment Videos
Interpreting conformational effects in protein nano-ESI-MS spectra
Maria Samalikova1, Irena Matecko, Norbert Müller
1Institute of Chemistry, Johannes Kepler University, Altenbergerstrasse 69, 4040 Linz, Austria.
Analytical and Bioanalytical Chemistry
|December 10, 2003
Summary
Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) reveals protein unfolding due to gas flow and pH. Protein charge-state distributions (CSD) are characteristic of conformation, not directly affected by pH or solvent surface tension.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Physical Chemistry
Background:
- Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is a powerful tool for analyzing protein conformations.
- Understanding the influence of experimental parameters on protein charge-state distributions (CSD) is crucial for accurate interpretation of nano-ESI-MS data.
- Protein stability can be affected by factors such as pH, solvent composition, and instrumental settings.
Purpose of the Study:
- To investigate equilibrium protein conformational transitions using nano-ESI-MS.
- To analyze the impact of instrumental settings, pH, and solvent surface tension on protein CSD.
- To determine the reliability of nano-ESI-MS for studying protein conformational stability under various conditions.
Main Methods:
- Utilized nano-ESI-MS to study conformational changes in cytochrome c, myoglobin, ubiquitin, and lysozyme.
- Manipulated instrumental settings, including nitrogen gas flow rates.
- Adjusted solution pH using hydrochloric acid, formic acid, and acetic acid, and varied solvent surface tension.
Main Results:
- High nitrogen gas flow rates can induce protein unfolding, particularly when combined with acidification.
- Protein CSD are characteristic of their native or unfolded conformations and are not directly altered by significant pH changes with HCl.
- Acidification with acetic or formic acid can induce unfolding, likely due to increased solvent hydrophobicity, but CSD are not limited by surface tension.
- Observed similar CSD for proteins in solutions with varying surface tensions, suggesting a potential lack of dependence on this parameter.
Conclusions:
- Nano-ESI-MS can accurately describe equilibrium protein unfolding transitions when instrumental parameters are optimized.
- Protein CSD are robust indicators of protein conformation, largely independent of pH and solvent surface tension under tested conditions.
- The influence of acetic acid on protein CSD may be less significant than previously assumed, or its effect on droplets is limited.