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Aging and Oxidative Damage to Mitochondrial Proteins

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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Aging and Oxidative Damage to Mitochondrial Proteins

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

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

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

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

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

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

  8. Thioredoxin as an in vitro model

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

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

  11. N-terminus b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 C-terminus I-I-H-L-T-D-D-S-F-D-T-D-V-L-K y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 100 HNE-ARP I-I L T D D S F D T-D V H b2 227.1 b3 833.5 % b4 946.6 b5 1048 b7 1278 b8 1365 b6 1163 b10 1627 b9 1512 b12 1843 b13 1942 0 200 400 600 800 1000 1200 1400 1600 1800 m/z MS/MS of thioredoxin peptides

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

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

  14. MS of unlabeled peptides 100 MIAPILDEIADEYQGK 1805.9 % 0 799.0 1441.8 2084.6 2727.4 3370.2 4013.0 m/z

  15. HNE-ARP HNE-ARP IIHLTDDSFDTDVLK SDKIIHLTDDSFDTDVLK 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

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

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