| Spatially Resolved Protein Hydrogen Exchange Measured by Subzero-Cooled Chip-Based Nanoelectrospray Ionization Tandem Mass Spectrometry. | |
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MedLine Citation:
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PMID: 22536891 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the back-exchange at quench conditions to very low levels. For example, only 4% and 6% deuterium loss for fully deuterated ubiquitin and β(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully optimized mass spectrometric conditions where gas-phase hydrogen scrambling is negligible. Our results show that the known dynamic behavior of ubiquitin in solution is accurately reflected in the deuterium contents of the fragment ions. |
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Authors:
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Sabine Amon; Morten B Trelle; Ole N Jensen; Thomas J D Jørgensen |
Publication Detail:
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Type: JOURNAL ARTICLE Date: 2012-4-26 |
Journal Detail:
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Title: Analytical chemistry Volume: - ISSN: 1520-6882 ISO Abbreviation: - Publication Date: 2012 Apr |
Date Detail:
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Created Date: 2012-4-27 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0370536 Medline TA: Anal Chem Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
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Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, DK-5230 Odense M, Denmark. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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