Subzero Celsius separations in three-zone temperature controlled hydrogen deuterium exchange mass spectrometry
Thomas E Wales1, Keith E Fadgen2, Michael J Eggertson2
1Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115, United States.
Journal of Chromatography. A
|June 10, 2017
Summary
Subzero temperature separations in hydrogen deuterium exchange mass spectrometry (HDX MS) significantly improve deuterium recovery for protein analysis. This method enhances protein conformational landscape studies by enabling longer gradients and higher peak capacity.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Structural Biology
Background:
- Hydrogen deuterium exchange mass spectrometry (HDX MS) is crucial for studying protein conformational dynamics.
- Maintaining deuterium labels during chromatography requires low temperatures (typically 0°C) to prevent exchange and solvent freezing.
- Existing methods face limitations in deuterium recovery and analytical timescale.
Purpose of the Study:
- To develop and validate a modified HDX MS system for peptide separations at subzero temperatures.
- To assess the impact of subzero separations on deuterium recovery and chromatographic performance.
- To enable enhanced analysis of complex protein systems through improved deuterium retention.
Main Methods:
- Modification of a Waters nanoACQUITY HDX manager with a third temperature-controlled zone for subzero separations.
- Integration of Peltier cooling for the chromatography column and trapping system.
- Use of 35% methanol post-digestion to prevent mobile phase freezing.
- Separation of peptides using conventional HPLC columns at temperatures down to -20°C.
Main Results:
- Subzero separations (-20°C) demonstrated significant increases in deuterium recovery for model peptides and proteins compared to 0°C.
- Chromatographic efficiency remained largely unchanged with subzero temperatures using standard HPLC columns.
- The enhanced deuterium recovery allows for expanded gradient programs, increasing peak capacity.
Conclusions:
- Subzero temperature separations are a viable and effective strategy to improve deuterium retention in HDX MS.
- This technical advancement facilitates the analysis of larger, more complex proteins and systems.
- The modified system offers a promising approach for more comprehensive protein structural studies.
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