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
oxidative stress
mitochondrial dysfunction
myocardial aging
 Molecular mechanisms largely unknown
 Finding which proteins are damaged can provide
an insight into cardiac aging on the molecular
level
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
OH
O
= Young Rats
4-hydroxy-2-nonenal (4-HNE)
= 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
OH
 Adduction assumed to
be mostly on Cys, Lys,
His (most
nucleophilic)
O
4-HNE
H
N
H2N
SH
N
Protein
Protein
Aldehyde functionality group
OH
O
Lysine
OH
O
C5H11
S
Histidine
Protein
Cysteine
OH
O
C5H11
O
C5H11
HN
Protein
N
N
Protein
4-hydroxy-2-nonenal (4-HNE)
OH
Protein
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
Aldehyde functionality group
lipid adducts)
OH
O
O
HICAT
HICAT
IV
+ HICAT
HN
IV
Biotin
4-hydroxy-2-nonenal (4-HNE)
polyethylene
oxide
NH
H
N
S
label
O
O
O
13C
HN
O
CH2
CH2
hydrazide
NH
NH2
O
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
HICAT
BN-PAGE
IV
4˚C
Western blot
Develop
w/ Hrp
film
HICAT
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
H
S
HN
100
O
H
N
m/z 375
O
O
O
N H
H
NH
m/z 270
Relative Intensity (%)
MH+
930
O
m/z 457
375
75
O
m/z 489
HN
N
457
50
S
m/z 801
H
N
HN
O
H2 N
OH
O
O
m/z 855
25
O
OH
270
489
0
100
200
300
400
500
m/z
623.4
600
801 855
700
800
MS/MS of 12C-HICAT-DDE-Glutathione
900
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
Linoleic Acid
COOH
COOH
HOO
COOH
COOH
13-HPODE
COOH
COOH
9-HPODE
COOH
COOH
COOH
OOH
COOH
COOH
COOH
O
HOO
OOH
O
O
OOH
OH
O
O
OOH
O
OH
O
O
OOH
O
HOO
2,4-DDE
O
OH
O
13-KODE
4-HPNE
13-HODE
O
9-KODE
Acrolein
4-ONE
4-HNE
9-HODE
Experimental:
Biocytin-Hydrazide Label
Shorter chain length vs. HICAT
Commercially available
O
HN
NH
H
N
S
O
NHNH2
O
Biocytin-Hydrazide
NH2