1. 2012 YCU Science Summer Program Lecture #6 Proteomic Approach to Protein Alterations Implicated in Aging and Geriatric Diseases Tosifusa Toda Yokohama City University Advanced Medical Research Center 2012.08.15

2. Genome, Transcriptome and Proteome Genome: entire set of genes (ca. 23,000 genes/individual) DNA Folding Post-translational modification PDB Proteome: > 10,000 proteins/cell Transcriptome: a set of mRNA (> 3,000 messages/cell) Transcription mRNA Translation Ribosome tRNA Polypeptides mRNA Cell Individual

3. Posttranslational modification of protein 1. Physiological (enzymatic) modification 1.1. Phosphorylation 1.2. Glycosylation 1.3. Acetylation 1.4. Methylation 1.5. Ubiquitination 1.6. Deimination (Citrullination)

4. Posttranslational modification of protein 1. Physiological (enzymatic) modification 2. Non-physiological (non-enzymatic) modification 1.1. Phosphorylation 1.2. Glycosylation 1.3. Acetylation 1.4. Methylation 1.5. Ubiquitination 1.6. Deimination (Citrullination) 2.1. Oxidation 2.2. Glycation

5. Posttranslational modification of protein 1. Physiological (enzymatic) modification 1-1. Phosphorylation 2-1. Oxidation 2-2. Glycation Met Gly Asn Leu Ser Thr Lys Tyr Arg Cys Met Ala P P P

6. Posttranslational modification of protein 1. Physiological (enzymatic) modifications 1-2. Glycosylation

7. Posttranslational modification of protein 1. Physiological (enzymatic) modifications 1-3. Acetylation Interact with “Bromodomain” of DNA binding proteins

8. Posttranslational modification of protein 1. Physiological (enzymatic) modifications 1-4. Methylation Arginine

9. Posttranslational Modification of Protein 1. Physiological (enzymatic) modifications 2. Non-physiological (non-enzymatic) modifications 1.6. Deimination (Citrullination) 2-1. Oxidation 2-2. Glycation

10. Multiple posttranslational modifications (A typical example: Nucleosomal Histone H3) Citrulline

11. Oxidative stress: The most suspicious cause of aging

12. Oxidative Protein Modifications Implicated in Aging Carbonylation

13. Oxidative Protein Modifications Implicated in Aging Tyrosine Nitration Methionine Sulfoxidation

14. How do we analyze so many proteins comprehensively including various post-translational modifications? Proteomic Approach Proteomics: A strategy for analyzing many proteins in a sample at a time 1. Two-dimensional gel electrophoresis-based Proteomics 2. Liquid chromatography-based Shot-gun Proteomics

15. Protein mixture in a sample Mass spectrometry Database search Identification 、 PTM analysis Image analysis Spot picking 1. Two-dimensional gel electrophoresis-based Proteomics Separation by 2-DE In-gel digestion

16. Aging-related Protein Alterations Detected in Mouse Hippocampus by 2-DE-Based Proteome Analysis

17. Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

18. Oxidation of Calmodulin Detected by Spectrometry

19. Aging-Related Increase in Methionine Sulfoxide Level in Mouse Hippocampus

20. Native recombinant calmodulin Oxidized calmodulin Kd=34.67μM Kd=178.89 μM Functional impairment in oxidized Calmodulin

21. Multiple Functions of CaM

22. Development / maturation Genome DNA (ca.23,000 genes) mRNA (ca. 3,000 messages/cell) Primary translation products (ca. 3,000 proteins/cell ） Post-translationally modified proteins Proteomic phenotype Genetic background Functional network of normal proteins Oxidative Stress Implicated in Aging and Geriatric Diseases Epigenetic regulation Translation Transcription Enzymatic modification Senescence accelerating factor Oxidative Stress Oxidized proteins (Ox calmodulin etc) Aging Aging-related physical deterioration Geriatric diseases Oxidative modification Interfering Reductase