1. Proteomics of Protein Glutathionylation in Mitochondria of an Aging Heart Jessica Page Mentor: Dr. Claudia Maier Department of Chemistry

2. Mitochondria

3. Protein Glutathionylation ROS create an imbalance between glutathione (GSH) and glutathione disulfide (GSSH) ROS create an imbalance between glutathione (GSH) and glutathione disulfide (GSSH) Glutathionylation: Modification of proteins with GSSG

4. + Protein-SH + GSSGProtein-S-SG + GSHGlutathionylation 2 GSHGSSG

5. Goal Does damage to mitochondria as a result of protein oxidation and glutathionylation contribute to the loss of mitochondrial function in the aging heart?

6. Specific Aims Personal aims Personal aims

7. BiotinLinker Functional Group Affinity Attachment Affinity Chromatography Gel Electrophoresis Western Blot Streptavidin Probe Components and Functionalities

8. Probe Synthesis + PBSA Cys-ICAT dimer

9. 117011801190 m/z 0 100 % 1181.5272 1182.5375 1183.5480 1184.5430 Cys-ICAT (dimer) 1180.527 JMP004 MH+

10. Cys-ICAT dimer reduction

11. 580585590595600605610 m/z 0 100 % 592.2659 593.2763 594.2650 595.5048 MH + Cys-ICAT 591.2659 JMP004 Reduction using TCEP

12. + Title

13. Proof of Concept: Probe + GSSG

14. SDS-Page/Blot/Spot

15. Step 1: Protein Separation: 2D Gel Step 2: Excise Gel Spot Step 3: Proteolyze/extract peptides Step 4: Mass Spectrometry Peptide Mass Fingerprint Sequence Tag Step 5: Protein Identity (Database Search) Future: Combination of MS-Based Proteomic and MY probe Creatine Kinase