Shifts in protein charge state distributions with varying redox reagents in nanoelectrospray triple quadrupole mass spectrometry



The influence of a number of redox reagents on the charge state distribution in nanoelectrospray mass spectrometry was examined using cytochrome c and ubiquitin. The redox active species investigated were: 1,4-benzoquinone, quinhydrone, tetracyanoquinodimethane (TCNQ), hydroquinone, and ascorbic acid. The redox active species was mixed with the protein sample before injection into the nanoelectrospray emitter, and mass spectra were acquired using a triple quadrupole mass spectrometer. Under the same experimental conditions, the charge state distribution of cytochrome c was observed to shift from a weighted average charge state of 14.25 (in the absence of redox species) to 7.10 in the presence of 1,4-benzoquinone. When quinhydrone was mixed with cytochrome c, the charge state distribution of the protein also shifted to lower charge states (weighted average charge state = 9.43), indicative of less charge state reduction for quinhydrone than with 1,4-benzoquinone. Addition of the redox reagent had little effect on the conformation of cytochrome c, as indicated by far ultraviolet circular dichroism spectra. In contrast, the reagents TCNQ, hydroquinone, and ascorbic acid exhibited negligible effects on the observed charge state distribution of the protein. The differing results for these redox reagents can be rationalized in terms of the redox half reactions involving these species. The results observed with ubiquitin upon adding quinhydrone were analogous to those observed with cytochrome c.


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Copyright information

© American Society for Mass Spectrometry 2004

Authors and Affiliations

  • Cheng Zhao
    • 1
  • Troy D. Wood
    • 1
    • 2
    • 3
  • Stanley Bruckenstein
    • 1
  1. 1.Department of ChemistryState University of New York at BuffaloBuffaloUSA
  2. 2.Department of Structural BiologyThe State University of New York at BuffaloBuffalo
  3. 3.Department of Molecular and Cellular BiophysicsRoswell Park Cancer InstituteBuffalo

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