Document Detail


Subzero temperature chromatography for reduced back-exchange and improved dynamic range in amide hydrogen/deuterium exchange mass spectrometry.
MedLine Citation:
PMID:  23025328     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified.
Authors:
John D Venable; Linda Okach; Sanjay Agarwalla; Ansgar Brock
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-10-19
Journal Detail:
Title:  Analytical chemistry     Volume:  84     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-06     Completed Date:  2013-11-26     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  9601-8     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amides / chemistry*
Amino Acid Sequence
Buffers
Chromatography, Reverse-Phase / methods*
Deuterium Exchange Measurement / methods*
Humans
Mass Spectrometry / methods*
Molecular Sequence Data
Peptides / chemistry
Temperature*
Time Factors
Grant Support
ID/Acronym/Agency:
U54 GM094586/GM/NIGMS NIH HHS; U54 GM094586/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Amides; 0/Buffers; 0/Peptides
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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