Aging and Oxidative Damage to Mitochondrial Protein Subunits of Complex IV

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. = Young Rats = Old Rats 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 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), 1064-1072

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

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

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

8. HICAT HICAT HICAT IV 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 BN-PAGE 4˚C Develop Western blot film w/ Hrp

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

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. Complex I Complex V Complex III Complex IV Complex II Results:ETC Proteins in Blue Native-PAGE 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. MH+ 930 100 375 75 Relative Intensity (%) 457 50 25 270 489 623.4 801 855 0 100 200 300 400 500 600 700 800 900 m/z MS/MS of 12C-HICAT-DDE-Glutathione Results:HICAT Breakdown Detected • Small peaks at 375 and 457 detected

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