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Increased protein structural resolution from diethylpyrocarbonate-based covalent labeling and mass spectrometric
Yuping Zhou1, Richard W Vachet
1Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
Journal of the American Society for Mass Spectrometry
|February 3, 2012
Summary
Shortening protein digestion times after diethylpyrocarbonate (DEPC) labeling significantly enhances protein structural information. This method increases DEPC-modified residues, improving protein structural resolution for mass spectrometry analysis.
Area of Science:
- Biochemistry
- Structural Biology
- Analytical Chemistry
Background:
- Covalent labeling combined with mass spectrometry is increasingly used to study protein 3D structures.
- Diethylpyrocarbonate (DEPC) is a promising reagent for probing protein residues due to its reactivity and single reaction product.
- Previous studies indicated DEPC modifications can be labile, potentially limiting structural insights.
Purpose of the Study:
- To investigate the extent of DEPC label loss on various amino acid residues.
- To determine methods for increasing the number of DEPC-modified residues for enhanced structural analysis.
- To improve the protein structural resolution obtained via DEPC covalent labeling.
Main Methods:
- Proteins were covalently labeled with DEPC.
- Label stability was assessed under varying protein digestion times.
- Short proteolytic digestions (2 h) using immobilized chymotrypsin or Glu-C were employed.
- Mass spectrometry was used to analyze DEPC-modified residues.
Main Results:
- DEPC label loss is significant for Ser, Thr, Tyr, and His residues, especially with long digestion times.
- Decreasing digestion time between labeling and mass spectrometry substantially increases modified residues.
- Short digestion times increased DEPC modification percentages for cytochrome c (44% to 67%), myoglobin (35% to 81%), and β-2-microglobulin (76% to 95%).
- Short digestion times reduced the average distance between modified residues, enhancing structural resolution.
Conclusions:
- Label lability of DEPC is more widespread than previously recognized, particularly with extended digestion.
- Optimizing digestion time is crucial for maximizing DEPC labeling efficiency and protein structural information.
- Short proteolytic digestions offer a robust strategy to enhance protein structural resolution using DEPC labeling and mass spectrometry.
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