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Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry
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Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Published on: June 15, 2021

Protein surface mapping using diethylpyrocarbonate with mass spectrometric detection.

Vanessa Leah Mendoza1, Richard W Vachet

  • 1Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.

Analytical Chemistry
|March 15, 2008
PubMed
Summary

Diethylpyrocarbonate (DEPC) is now a more reliable protein surface probe, modifying Ser and Thr residues alongside His and Tyr. Mass spectrometry monitoring ensures protein structural integrity during labeling.

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Glycopeptide Capture for Cell Surface Proteomics
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Area of Science:

  • Biochemistry
  • Structural Biology
  • Analytical Chemistry

Background:

  • Diethylpyrocarbonate (DEPC) is a covalent labeling reagent used to probe protein surface structure.
  • Previous applications were limited by the residues DEPC could modify and potential structural perturbations.
  • Mass spectrometry offers high sensitivity and specificity for detecting chemical modifications.

Purpose of the Study:

  • To enhance the reliability and information content of DEPC as a protein surface structure probe.
  • To expand the range of residues modified by DEPC.
  • To establish a mass spectrometry-based method for ensuring protein structural integrity during labeling.

Main Methods:

  • Utilized myoglobin, cytochrome c, and beta-2-microglobulin as model systems.
  • Employed mass spectrometry to monitor DEPC modification extent as a function of reagent concentration (dose-response plots).
  • Compared DEPC labeling results with known X-ray crystal structures.

Main Results:

  • Demonstrated that DEPC modifies Ser and Thr residues in addition to His and Tyr.
  • Showed that dose-response plots reliably identify and avoid protein structural perturbations.
  • Identified solvent accessibility and nearby charged residues as key factors influencing modification rates.
  • Determined that Cu(II) binds His31 in beta-2-microglobulin, altering local residue accessibility.

Conclusions:

  • DEPC, when used with mass spectrometry and dose-response analysis, is a powerful and reliable tool for protein surface mapping.
  • The expanded residue modification range increases DEPC's utility, covering approximately 25% of an average protein sequence.
  • This method provides quantitative reactivity data and insights into protein structural changes upon ligand binding.