Protocol for the thermodynamic analysis of some proteins using an H/D exchange- and mass spectrometry-based technique.
SUPREX (stability of unpurified proteins from rates of H/D exchange) is a new H/D exchange- and mass spectrometry-based technique for the measurement of protein folding free energies (i.e., DeltaG values) and protein folding m values (i.e., deltaDeltaG/delta[denaturant]). Robust protocols for the acquisition and analysis of SUPREX data have been established and shown to be useful for the analysis of a number of different protein systems. Here we report on the SUPREX behavior of a special class of proteins that are not amenable to conventional SUPREX analyses using previously established protocols. This class of proteins includes protein systems that require an extended time to reach a folding/unfolding equilibrium in chemical denaturant-induced equilibrium unfolding experiments. As part of this work we use ubiquitin as a model system to highlight the complications that can arise in the conventional SUPREX analysis of such protein systems, and we describe a modified SUPREX protocol that can be used to eliminate these complications.
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Related Subject Headings
- Ubiquitin
- Time
- Thermodynamics
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Proteins
- Protein Folding
- Protein Denaturation
- Deuterium Exchange Measurement
- Analytical Chemistry
- 4004 Chemical engineering
Citation
Published In
DOI
EISSN
ISSN
Publication Date
Volume
Issue
Start / End Page
Related Subject Headings
- Ubiquitin
- Time
- Thermodynamics
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Proteins
- Protein Folding
- Protein Denaturation
- Deuterium Exchange Measurement
- Analytical Chemistry
- 4004 Chemical engineering