| Increased protein structural resolution from diethylpyrocarbonate-based covalent labeling and mass spectrometric detection. | |
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MedLine Citation:
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PMID: 22298289 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for cytochrome c, and 9 to 8 Å for β-2-microglobulin. |
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Authors:
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Yuping Zhou; Richard W Vachet |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural |
Journal Detail:
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Title: Journal of the American Society for Mass Spectrometry Volume: 23 ISSN: 1879-1123 ISO Abbreviation: J. Am. Soc. Mass Spectrom. Publication Date: 2012 Apr |
Date Detail:
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Created Date: 2012-04-20 Completed Date: 2012-06-28 Revised Date: 2013-05-20 |
Medline Journal Info:
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Nlm Unique ID: 9010412 Medline TA: J Am Soc Mass Spectrom Country: United States |
Other Details:
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Languages: eng Pagination: 708-17 Citation Subset: IM |
Affiliation:
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Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Diethyl Pyrocarbonate / chemistry* Mass Spectrometry / methods* Molecular Sequence Data Protein Conformation Proteins / chemistry* |
| Grant Support | |
ID/Acronym/Agency:
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R01 GM075092/GM/NIGMS NIH HHS; R01 GM075092-07/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Proteins; 1609-47-8/Diethyl Pyrocarbonate |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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