Abstract/Details

Using metal catalyzed oxidation reactions and mass spectrometry as a reliable method for determining metal binding-sites in proteins


2006 2006

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Abstract (summary)

The combination of metal catalyzed oxidation (MCO) reactions and mass spectrometry (MS) is a relatively new method of determining the coordination structure of metalloproteins. The sensitivity of the MCO/MS method gives it significant potential for the study of protein structure. The method takes advantage of Fenton-type oxidation reactions occurring at protein-bound transition-metal ions. These reactions produce reactive oxygen species that modify metal binding residues, and the modified residues are identified using the peptide sequencing ability of MS.

This dissertation focuses on increasing the reliability and versatility of the MCO/MS method as a tool for studying metal-protein interactions. The importance of ascorbate concentration in controlling the oxidation yield and specificity of the MCO reactions has been established, and sodium persulfate has been identified as a specific and potent oxidant. Greater insight into the role of ascorbate has allowed us to develop a new "detuned" version of MCO/MS method that can oxidize residues beyond the metal-binding site, making the method more sensitive to minor changes in protein structure that might occur upon metal binding to a protein. Application of the detuned method to Cu binding of β-2-microglobulin suggests some possible structural changes caused by metal binding that might provide insight into the amyloid formation of this protein.

The versatility of the MCO/MS approach has been expanded by, improving the speed of the method and extending its application to other metals. In an effort to better study dynamic systems, microwave irradiation has been used to decrease the time required for MCO reactions by a factor of 10. The utility of the MCO/MS method for the study of non-Cu transition metal systems has been illustrated by determining suitable MCO reaction conditions for peptides that bind Mn, Fe, Co and Ni and a whole protein, Nickel superoxide dismutase.

Finally, we have studied the effect of amino acid oxidation on the dissociation patterns of peptide ions during tandem MS (MS/MS) experiments and the identification of binding residues. We have found that histidine oxidation changes the peptides dissociation pattern. We demonstrate that an N-terminal derivatization method can be used to simplify the MS/MS interpretation in such circumstances.

Indexing (details)


Subject
Analytical chemistry;
Biochemistry
Classification
0486: Analytical chemistry
0487: Biochemistry
Identifier / keyword
Pure sciences, Mass spectrometry, Metal-catalyzed oxidation, Oxidation, Proteins
Title
Using metal catalyzed oxidation reactions and mass spectrometry as a reliable method for determining metal binding-sites in proteins
Author
Bridgewater, Juma D.
Number of pages
185
Publication year
2006
Degree date
2006
School code
0118
Source
DAI-B 67/11, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9780542977121
Advisor
Vachet, Richard W.
University/institution
University of Massachusetts Amherst
University location
United States -- Massachusetts
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3242308
ProQuest document ID
305309008
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/305309008
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