Aging and oxidative damage to mitochondrial proteins
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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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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


Oxygen radicals and aging

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


Oxidative phosphorylation

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


Formation of lpo products

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)


Oxylipid adduction to proteins

electrophilic C-3

Oxylipid adduction to proteins

Cysteine

Histidine

4-HNE

Lysine


Oxidative phosphorylation1

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


Probing for damaged proteins

biotin

hydroxylamine

Aldehyde functionality group

ARP

(Aldehyde-reactive probe)

4-HNE-adducted protein

Probing for damaged proteins


Thioredoxin as an in vitro model

Thioredoxin as an in vitro model


Thioredoxin as an in vitro model experimental approach

+ HNE & ARP

+ Trypsin

Thioredoxin peptides

Thioredoxin as an in vitro model:Experimental Approach

Thioredoxin


Ms of arp labeled thioredoxin peptides

[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


Ms ms of thioredoxin peptides

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


Affinity chromatography

= ARP

biotin

hydroxylamine

ARP

(Aldehyde-reactive probe)

Affinity chromatography

Avidin bead column


Affinity chromatography cont d

= ARP

Affinity chromatography (cont’d)

Avidin bead column


Ms of unlabeled peptides

MS of unlabeled peptides

100

MIAPILDEIADEYQGK

1805.9

%

0

799.0

1441.8

2084.6

2727.4

3370.2

4013.0

m/z


Ms of labeled peptides

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


Future work

Future work

  • Maximize recovery of material from beads

  • Repeat with labeled mitochondrial samples

  • Find which and where proteins have been adducted


Acknowledgements

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


Questions

Questions?


Avidin immobilized beads

Avidin-immobilized beads

acrylamide

monomeric avidin

azlactone linker


Affinity chromatography1

biotin

IV

ARP

LPO

biotin

avidin

avidin

avidin

avidin

avidin

irreversible bond

reversible bond

ARP-labeled Complex IV

Affinity chromatography


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