1. Aging and Oxidative Damage to Mitochondrial Proteins
Tony Tong
Dr. Claudia Maier
Dr. Fred Stevens
Department of Chemistry
Department of Pharmaceutical Sciences
Department of Biochemistry and Biophysics
Oregon State University
August 26, 2005
HHMI Summer 2005 Fellowship Program

2. Oxygen radicals and aging
ROS (H2O2, ·O2-, ·OH)
Oxidative damage
Heart disease and aging
Source: Core81/chap3.html
Overall goal: Characterize oxidative damage to mitochondrial proteins

3. Oxidative Phosphorylation
Cell cytosol
Outer membrane
Intermembrane
space
Inner membrane
Matrix
Oxidative Phosphorylation H+ H+ H+ e-
III I IV V
O2 + H+ e- 4
+ 4
2 H2O
ADP + Pi
H2O2, ·O2-, •OH
ATP
NADH
Adapted from Cooper, G. and Hausman, R., Cell: A Molecular Approach, 2003

4. Formation of LPO products
Linoleic acid
ROS (H2O2, ·O2-, ·OH)
Aldehyde functionality group
4-hydroxy-2-nonenal (4-HNE)
4-oxo-2-nonenal (4-ONE)

5. Oxylipid adduction to proteins
Cysteine
electrophilic C-3
Histidine
4-HNE
Lysine

6. Oxidative Phosphorylation
Cell cytosol
Outer membrane
Intermembrane
space
Inner membrane
Matrix
Oxidative Phosphorylation H+ H+ H+ e-
III I IV V
O2 + H+ e- 4
+ 4
2 H2O
ADP + Pi
H2O2, ·O2-, •OH
ATP
NADH
Adapted from Cooper, G. and Hausman, R., Cell: A Molecular Approach, 2003

7. Probing for damaged proteins
ARP
(Aldehyde-reactive probe)
hydroxylamine
biotin
4-HNE-adducted protein
Aldehyde functionality group

8. Thioredoxin as an in vitro model

9. Thioredoxin as an in vitro model: Experimental Approach
+ HNE & ARP
Thioredoxin
+ Trypsin
Thioredoxin peptides

10. MS of ARP-labeled thioredoxin peptides
734.4
HNE-ARP
[IIHLTDDSFDTDVLK]3+
+ HNE
630.3
HNE
[IIHLTDDSFDTDVLK]3+
577.9
[IIHLTDDSFDTDVLK]3+
m/z

11. MS/MS of thioredoxin peptides
I-I-H-L-T-D-D-S-F-D-T-D-V-L-K b1 b2 b3 b4 b5 b6 b7 b8 b9
b10
b11
b12
b13
b14
y14
y13
y12
y11
y10 y9 y8 y7 y6 y5 y4 y3 y2 y1
N-terminus
C-terminus
200
400
600
800
1000
1200
1400
1600
1800
100 %
m/z
I-I
HNE-ARP H L T D S F
T-D V b2
227.1 b3
833.5 b7
1278 b6
1163 b8
1365 b9
1512
b10
1627
b12
1843
b13
1942 b4
946.6 b5
1048

12. Affinity chromatography
= ARP
ARP
(Aldehyde-reactive probe)
hydroxylamine
biotin
Avidin bead column

13. Affinity chromatography (cont’d)
= ARP
Avidin bead column

14. MS of unlabeled peptides
100
MIAPILDEIADEYQGK
1805.9 %
799.0
1441.8
2084.6
2727.4
3370.2
4013.0
m/z

15. MS of labeled peptides IIHLTDDSFDTDVLK SDKIIHLTDDSFDTDVLK 2201.1
100
IIHLTDDSFDTDVLK
HNE-ARP %
SDKIIHLTDDSFDTDVLK
HNE-ARP
2201.1
2531.3
799.0
1441.8
2084.6
2727.4
3370.2
4013.0
m/z

16. Future work Maximize recovery of material from beads
Repeat with labeled mitochondrial samples
Find which and where proteins have been adducted

17. Acknowledgements The labs of: Dr. Claudia Maier Dr. Fred Stevens
Dr. Mike Schimerlik
Dr. Emily Ho
EHSC Mass Spectrometry Core Facility
Dr. Kevin Ahern
Dr. Chris Mathews
Howard Hughes Medical Institute
Sacrificed rats

18. Questions?

19. Avidin-immobilized beads
acrylamide
monomeric avidin
azlactone linker

20. Affinity chromatography
ARP-labeled Complex IV
ARP
LPO IV
avidin
biotin
reversible bond
avidin
biotin
irreversible bond