Aging and Oxidative Damage to Mitochondrial Proteins

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
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Oxygen radicals and aging
Source http//onlinetc.its.brooklyn.cuny.edu/
Core81/chap3.html
Overall goal Characterize oxidative damage to
mitochondrial proteins
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Oxidative Phosphorylation
V
IV
ATP
Adapted from Cooper, G. and Hausman, R., Cell A
Molecular Approach, 2003
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Formation of LPO products
Linoleic acid
ROS (H2O2, O2-, OH)
4-hydroxy-2-nonenal (4-HNE)
4-oxo-2-nonenal (4-ONE)
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Oxylipid adduction to proteins
Cysteine
Histidine
4-HNE
Lysine
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Oxidative Phosphorylation
V
IV
ATP
Adapted from Cooper, G. and Hausman, R., Cell A
Molecular Approach, 2003
7
Probing for damaged proteins
8
Thioredoxin as an in vitro model
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Thioredoxin as an in vitro modelExperimental
Approach
Thioredoxin
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MS of ARP-labeled thioredoxin peptides
m/z
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MS/MS of thioredoxin peptides
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Affinity chromatography
Avidin bead column
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Affinity chromatography (contd)
Avidin bead column
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MS of unlabeled peptides
100
MIAPILDEIADEYQGK
1805.9

0
799.0
1441.8
2084.6
2727.4
3370.2
4013.0
m/z
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MS of labeled peptides
100

2201.1
2531.3
0
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

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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

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Questions?
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Avidin-immobilized beads
acrylamide
monomeric avidin
azlactone linker
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Affinity chromatography