This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Assignment of oxidation sites for hydroxyl radical footprinting experiments is done by means of tandem mass spectrometry, where the oxidized peptide is activated in the mass spectrometer and the resulting fragmentation pattern is compared to a computed theoretical fragmentation pattern, and the differences analyzed to determine where the oxidation occurred. However, the oxidation of side chains alters the fragmentation chemistry in ergodic fragmentation methods, often resulting in a smaller number of fragmentation products which makes absolute assignment of sites of oxidation difficult. We are attempting to utilize higher energies for ergodic fragmentation, coupled with scanning at lower mass to charge ratios, to try to find characteristic side chain ions and side chain losses that are diagnostic of oxidation at a particular amino acid residue, in order to simplify the assignment of oxidation sites. Another difficulty that often arises is the matter of oxidation isomers;a peptide on which oxidation occurs at two or more sites. Quantitation of the rates of oxidation on each site is not possible by simply quantitating the abundances of fragment ions, as oxidation affects the pattern of fragmentation itself in ergodic processes. Recent work has suggested that non-ergodic fragmentation methods such as electron capture dissociation (ECD) are insensitive to side chain oxidation, suggesting that fragment ion abundances may be useful for quantitating isomeric mixtures of oxidized peptides. We will be oxidizing model peptides with multiple oxidation sites and studying the abundances of fragment ions generated by ECD. These abundances will be compared with the abundances of oxidized and unoxidized amino acids generated by acid hydrolysis of the oxidized peptide in order to determine if quantitation by ECD fragment ion abundance is feasible.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR005351-22
Application #
8361802
Study Section
Special Emphasis Panel (ZRG1-IMST-A (40))
Project Start
2011-02-01
Project End
2012-01-31
Budget Start
2011-02-01
Budget End
2012-01-31
Support Year
22
Fiscal Year
2011
Total Cost
$35,432
Indirect Cost
Name
University of Georgia
Department
Type
Organized Research Units
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
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