2012 ycu science summer program lecture #6 proteomic approach to protein alterations implicated in...
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2012 YCU Science Summer Program2012 YCU Science Summer Program
Lecture #6
Proteomic Approach to Protein Alterations Implicated in Aging and Geriatric Diseases
Tosifusa Toda
Yokohama City UniversityAdvanced Medical Research Center
2012.08.15
Genome, Transcriptome and ProteomeGenome, Transcriptome and Proteome
Genome: entire set of genes(ca. 23,000 genes/individual)
DNA
Folding
Post-translationalmodification
PDB
Proteome: > 10,000 proteins/cell
Transcriptome: a set of mRNA (> 3,000 messages/cell)
Transcription
mRNA
Translation
Ribosome
tRNAPolypeptides
mRNA
Cell
Individual
Posttranslational modification of protein Posttranslational modification of protein
1. Physiological (enzymatic) modification
1.1. Phosphorylation1.2. Glycosylation1.3. Acetylation1.4. Methylation1.5. Ubiquitination1.6. Deimination (Citrullination)
Posttranslational modification of protein Posttranslational modification of protein
1. Physiological (enzymatic) modification
2. Non-physiological (non-enzymatic) modification
1.1. Phosphorylation1.2. Glycosylation1.3. Acetylation1.4. Methylation1.5. Ubiquitination1.6. Deimination (Citrullination)
2.1. Oxidation2.2. Glycation
Posttranslational modification of protein Posttranslational modification of protein
1. Physiological (enzymatic) modification
1-1. Phosphorylation
2-1. Oxidation2-2. Glycation
MetMet
GlyGly
AsnAsn
LeuLeu
SerSer
ThrThr
LysLys
TyrTyr
ArgArg
CysCys
MetMet
AlaAla
P
P
P
Posttranslational modification of proteinPosttranslational modification of protein1. Physiological (enzymatic) modifications
1-2. Glycosylation
Posttranslational modification of protein Posttranslational modification of protein
1. Physiological (enzymatic) modifications
1-3. Acetylation
Interact with “Bromodomain” of DNA binding proteins
Posttranslational modification of protein Posttranslational modification of protein
1. Physiological (enzymatic) modifications
1-4. Methylation
Arginine
Posttranslational Modification of Protein Posttranslational Modification of Protein1. Physiological (enzymatic) modifications
2. Non-physiological (non-enzymatic) modifications
1.6. Deimination (Citrullination)
2-1. Oxidation2-2. Glycation
Multiple posttranslational modifications(A typical example: Nucleosomal Histone H3)
Multiple posttranslational modifications(A typical example: Nucleosomal Histone H3)
Citrulline
Citrulline
Oxidative stress: The most suspicious cause of aging
Oxidative Protein Modifications Implicated in Aging
Carbonylation
Oxidative Protein Modifications Implicated in Aging
Tyrosine Nitration
Methionine Sulfoxidation
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
Protein mixturein a sample
Mass spectrometry
Database searchIdentification、
PTM analysis
Image analysis
Spot picking
1. Two-dimensional gel electrophoresis-based Proteomics
Separation by 2-DE
In-gel digestion
Aging-related Protein Alterations Detected in Mouse Hippocampus by 2-DE-Based Proteome
Analysis
Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry
Oxidation of Calmodulin Detected by Spectrometry
Aging-Related Increase in Methionine Sulfoxide Level in Mouse Hippocampus
Native recombinant calmodulin
Oxidized calmodulin
Kd=34.67μM
Kd=178.89 μM
Functional impairment in oxidized Calmodulin
Functional impairment in oxidized Calmodulin
Multiple Functions of CaM
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