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

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

Background: Oxidative Phosphorylation

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), 1064-1072

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

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

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)

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

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

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

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

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

Results: Blue Native-PAGE Gel Mouse heart mitochondria

Results: Blue Native-PAGE Blot

Results: Blue Native-PAGE Overlay HICAT-reactive spots

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

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

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

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

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

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!

Questions?

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

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

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

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

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

Background: LPO Product Formation

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