1. Aging and Oxidative Damage to Mitochondrial Protein Subunits of Complex IV
Tony Tong
Dr. Fred Stevens
Dr. Claudia Maier
Duane Mooney
Department of Chemistry
Department of Biochemistry and Biophysics
Oregon State University
September 1, 2004
HHMI Summer 2004 Fellowship Program

2. Background: Aging and Oxygen Radicals
Heart disease is the #1 cause of death over age 65
Large amounts of oxygen radicals are thought to contribute to:
protein oxidation
mitochondrial dysfunction
Molecular mechanisms largely unknown
Finding which proteins are damaged can provide an insight into cardiac aging on the molecular level
oxidative stress
myocardial aging

3. Background: Oxidative Phosphorylation

4. Background: Oxygen Radicals and Complex IV
Oxygen radicals can cause peroxidation of nearby lipids
These LPO products can then adduct to proteins, ie Complex IV
Studies have shown that as adduction of 4-HNE (a LPO product) increases, Complex IV activity decreases
= Young Rats
= Old Rats
Source: Suh, J. H., Heath, S. H., Hagen, T. M. (2003). Two subpopulations of mitochondria in the aging rat heart display heterogeneous levels of oxidative stress. Free Radical Biology & Medicine 35 (9),

5. Background: Adduction to Complex IV
Adduction assumed to be mostly on Cys, Lys, His (most nucleophilic)
Aldehyde functionality group

6. Background: Complex IV
aka cytochrome c oxidase
Comprised of 13 polypeptide subunits

7. Experimental: Labeling adducted Complex IV
Use rat and mouse heart mitochondria
HICAT label added
Label is aldehyde-specific (hydrazide binds to lipid adducts)
Aldehyde functionality group
Biotin
polyethylene
oxide
HICAT
13C label
hydrazide IV IV
+ HICAT
HICAT
(Hydrazide-functionalized Isotope-Coded Affinity Tag)

8. Experimental: Isolating adducted Complex IV
Membrane proteins separated by Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE)
Western blot with avidin-horseradish peroxidase (binds with biotin)
Adding H2O2 fluoresces Hrp
HICAT IV
BN-PAGE
4˚C
Western blot
Develop
w/ Hrp
film

9. Experimental: Isolating adducted Complex IV
Fluorescent spots overlaid with an identical gel
Matching spots are HICAT-reactive
All gel spots excised and digested with trypsin
Proteins extracted
Overlay film & 2nd gel
Gel pieces cut,
digested, extracted

10. Experimental: ID of adducted Complex IV proteins
Digested proteins analyzed by LC/Q-ToF* mass spectrometry
*Liquid Chromatography/Quadrupole-Time of Flight

11. Brief Background: Proteomics
Study of proteins
A certain sequence of amino acids defines a protein
Highly unlikely that 2 different protein fragments will have the same mass
ID of a sequence  ID of a protein

12. Experimental: ID of proteins
Q-ToF-MS/MS sequentially breaks up protein fragments further into amino acid residues
Analysis by MASCOT search engine identifies proteins

13. Results: Blue Native-PAGE Gel
Mouse heart mitochondria

14. Results: Blue Native-PAGE Blot

15. Results: Blue Native-PAGE Overlay
HICAT-reactive spots

16. Results: Mass spectrometry of peptides
15 fragment ions correspond to peptide ILYMMDEINNPVLTVK
Q-ToF-MS/MS Spectrum of a Complex IV Subunit II peptide

17. Results: Complex IV MASCOT Search Results
Subunits I, II, III, IV, Va, Vb, VIIa
7 of 13 subunits detected
Subunit VIc detected in rat heart mitochondria

18. Results: ETC Proteins in Blue Native-PAGE
Complex I
Complex V
Complex III
Complex IV
Complex II
HICAT-reactive spots

19. Results: Detecting HICAT adduction
None found in MASCOT searching
Concentration problem?

20. Experimental: Future approaches
2-D PAGE (2nd dimension w/SDS) for increased resolution
Affinity chromatography coupled with MS
Only adducted peptide fragments will be detected
Allows ID of adducted residues

21. Many thanks to… The labs of: Dr. Fred Stevens Dr. Claudia Maier
Dr. Tory Hagen
Dr. Emily Ho
Duane Mooney
Andy Larkin
Gretchen Clark-Scannell
Brian Arbogast
Dr. John Sowell
Dr. Kevin Ahern
Howard Hughes Medical Institute
OSU Undergraduate Research, Innovation, Scholarship, Creativity (URISC) Program
You for listening!

22. Questions?

23. Experimental: Affinity chromatography
Bead-immobilized avidin (commercially prepared)
Monomeric & multimeric forms (mono is weaker, biotin bond is reversible)
Irreversible sites blocked w/biotin
Glycine used to remove biotin from reversible sites
Proteins added, unbound protein washed away
Bound protein eluted with formic acid

24. Results: Affinity chromatography
Early work w/biotinylated insulin shows presence of protein in elution samples and none in nonbiotinylated protein washes
However, no labeled peptides found in Q-ToF
In MALDI, peptides were detected, but not enough to do MS/MS

25. Results: Affinity Chromatography Test Run
MALDI-ToF MS Spectrum of Insulin-Biotin

26. Results: HICAT Breakdown Detected
Small peaks at 375 and 457 detected
100
200
300
400
500
600
700
800
900
m/z 75 50 25
Relative Intensity (%)
270
375
457
489
623.4
801
930
MH+
855
MS/MS of 12C-HICAT-DDE-Glutathione

27. Results: The other bands?
Lower bands: Cytochrome c (ETC protein), actin, hemoglobin
Everywhere: Complex V subunits, citric acid cycle and ß-oxidation related proteins, myosin, albumin

28. Background: LPO Product Formation

29. Experimental: Biocytin-Hydrazide Label
Shorter chain length vs. HICAT
Commercially available
Biocytin-Hydrazide