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Proteomics of Protein Glutathionylation in Mitochondria of an Aging Heart

Proteomics of Protein Glutathionylation in Mitochondria of an Aging Heart. Jessica Page Mentor: Dr. Claudia Maier Department of Chemistry. Mitochondria. Protein Glutathionylation. ROS create an imbalance between glutathione (GSH) and glutathione disulfide (GSSH).

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Proteomics of Protein Glutathionylation in Mitochondria of an Aging Heart

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  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) Glutathionylation: Modification of proteins with GSSG

  4. 2 GSH GSSG Protein-SH + GSSG Protein-S-SG + GSH Glutathionylation +

  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

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

  8. Probe Synthesis + PBSA Cys-ICAT dimer

  9. MH+ Cys-ICAT (dimer)1180.527JMP004 1181.5272 100 1182.5375 % 1183.5480 1184.5430 0 m/z 1170 1180 1190

  10. Cys-ICAT dimer reduction

  11. Cys-ICAT591.2659JMP004 MH+ 592.2659 100 Reduction using TCEP % 593.2763 594.2650 595.5048 m/z 0 580 585 590 595 600 605 610

  12. Title +

  13. Proof of Concept: Probe + GSSG

  14. SDS-Page/Blot/Spot

  15. 4890 100 1777.0 90 80 Survey Scan Of Spot 22 70 % Intensity 60 50 40 1102.6 976.6 30 1648.9 20 2041.0 1913.0 2109.1 10 0 899 2200 1419 1679 1939 1159 Mass (m/z) Future: Combination of MS-Based Proteomic and MY probe Step 2: Excise Gel Spot Step 3: Proteolyze/extract peptides Step 4: Mass Spectrometry Step 1: Protein Separation: 2D Gel Peptide Mass Fingerprint Step 5: Protein Identity (Database Search) Sequence Tag Creatine Kinase

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