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Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Published on: November 29, 2013
False EX1 signatures caused by sample carryover during HX MS analyses
Jing Fang1, Kasper D Rand, Penny J Beuning
1Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115.
International Journal of Mass Spectrometry
|June 7, 2011
Summary
False EX1 kinetics in hydrogen exchange mass spectrometry (HX-MS) can be caused by peptide carryover. This study identifies carryover origins, explains its impact on HX-MS data, and presents an optimized washing protocol to eliminate it for reliable deuterium incorporation analysis.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Structural Biology
Background:
- Hydrogen exchange mass spectrometry (HX-MS) is a powerful technique for studying protein dynamics and structure.
- The EX1 kinetic model describes rapid deuterium incorporation upon simultaneous unfolding and solvent exposure of multiple residues.
- False EX1 kinetics can arise from experimental artifacts, complicating data interpretation.
Purpose of the Study:
- To investigate the phenomenon of false EX1 kinetics in HX-MS experiments.
- To identify peptide carryover as a significant contributor to false EX1.
- To develop and validate a method for eliminating peptide carryover in HX-MS.
Main Methods:
- Exploration of peptide carryover origins in chromatographic separations used in HX-MS.
- Analysis of mass spectra to understand how peptide carryover mimics EX1 kinetics.
- Development and testing of an optimized solvent washing protocol to remove residual peptides.
Main Results:
- Peptide carryover between chromatographic runs was identified as a primary cause of false EX1 signatures in HX-MS.
- A systematic series of solvent injections was optimized to effectively remove peptide carryover.
- The optimized washing protocol successfully eliminated carryover artifacts, enabling reliable deuterium incorporation measurements for proteins exhibiting high carryover.
Conclusions:
- Peptide carryover is a critical artifact that must be addressed for accurate HX-MS data interpretation.
- The developed washing protocol provides an effective solution for mitigating peptide carryover in HX-MS.
- This methodology ensures the reliability of deuterium incorporation studies, particularly for challenging protein samples.
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