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Electrospray Ionization (ESI) Mass Spectrometry01:12

Electrospray Ionization (ESI) Mass Spectrometry

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Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics
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Electrospray ionization-induced protein unfolding.

Hong Lin1, Elena N Kitova, Margaret A Johnson

  • 1Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2.

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|September 21, 2012
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Summary

Electrospray ionization mass spectrometry (ESI-MS) reveals protein unfolding in Clostridium difficile toxin B sub-fragments at low ionic strength. This structural change, observed in negative ion mode, is attributed to charge repulsion during the ESI process.

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Area of Science:

  • Biochemistry
  • Mass Spectrometry
  • Structural Biology

Background:

  • Clostridium difficile toxin B contains a carbohydrate-binding repeat region crucial for its function.
  • Sub-fragments of this region, like B3C, are studied to understand protein structure-stability relationships.
  • Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for analyzing protein structure in solution.

Purpose of the Study:

  • To investigate the solution conditions affecting the structure of a Clostridium difficile toxin B sub-fragment (B3C) using ESI-MS.
  • To explore the impact of ionic strength and charge on protein unfolding during ESI-MS analysis.
  • To compare the behavior of B3C with mutants (B4A, B4B) having reduced acidic residues.

Main Methods:

  • Electrospray ionization mass spectrometry (ESI-MS) in negative and positive ion modes.
  • Proton nuclear magnetic resonance (NMR) spectroscopy.
  • Circular dichroism (CD) spectroscopy.
  • Gel filtration chromatography.

Main Results:

  • ESI-MS in negative ion mode at low ionic strength (<80 mM) indicated unfolding of B3C, evidenced by higher charge states.
  • No unfolding was observed in positive ion mode or at higher ionic strengths (>10 mM) across various pH conditions.
  • NMR, CD, and gel filtration supported a predominantly folded state for B3C in neutral aqueous solutions with ionic strength >10 mM.
  • Mutants B4A and B4B showed smaller shifts to higher charge states at low ionic strength in negative ion mode ESI-MS.

Conclusions:

  • Protein unfolding of B3C observed in negative ion mode ESI-MS at low ionic strength is likely an artifact of the ESI process.
  • Coulombic repulsion between negatively charged residues and the droplet surface during ESI is proposed as the mechanism for unfolding.
  • The structural integrity of B3C is maintained in solution under physiological or higher ionic strength conditions.