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의학연구의실제, 03/19/2018

Overview
 Proteomics: definition and scopes
 Protein structure and function
 Workflow and technologies
 Applications

의과대학 약리학교실
김 학 림 Ph.D.

What & Why is Proteomics ?


 Different sciences describe many levels of biomolecular organization ---
What is Proteomics ?
but if used in isolation may give misleading inferences about the system !
 Differences Between Protein Chemistry and Proteomics

Protein Chemistry Proteomics

- Individual proteins - Complex mixtures

We Need the Integrated Science - Complete sequence analysis - Partial sequence analysis

- Emphasis on structure and function - Emphasis on identification

by database matching

- Structural biology - Systems biology


What is Proteomics ? What is Proteomics ?
 Three Kinds of Proteomics
 Proteomics employs an incredibly diverse
range of technologies including
- Expressional Proteomics
 Electrophoresis, Protein Chips, DNA Chips, SAGE
- Molecular biology  Mass Spectrometry, Microsequencing
- X-ray crystallography
- Functional Proteomics
- Chromatography  HT Functional Assays, Ligand Chips
- NMR spectroscopy  Yeast 2-hybrid, Deletion Analysis, Motif Analysis
- Protein Chemistry / Biochemistry
- Mass spectrometry - Structural Proteomics
 HT X-ray Crystallography / Modeling
- Computational biology  HT NMR Spectroscopy / Modeling

The Three Musketeers of Proteomics What is Proteomics ?


Integral to
understanding
biological systems is
the ability to discover
and measure changes
in the system

Science 291 (5507) 1221-1224, 2001

 Proteomics is an attempt to describe or explain biological state and


qualitative and quantitative changes of protein content of cells and
extracellular biological materials under different conditions to further
understand biological processes.
Levels of Biological Information From Genome to Biological Function

Proteomics vs Genomics Proteomics vs Genomics


Proteomics vs Genomics Human Genome Project

 Human Genes
- Estimated to be between 20,000 and 25,000 Francis Collins led
the National Human Genome Research Institute (NHGRI)
John Craig Venter, famous for his role in being one of
the first to sequence the human genome and for his
role in creating the first cell with a synthetic genome in
2010. Venter founded Celera Genomics, The Institute
for Genomic Research and the J. Craig Venter Institute,

- H. influenzae 1,709
- S. cerevisiae 6,241
- C. elegans 18,424
- D. melanogaster 13,601

 Human Proteome
- Estimated to be between 300,000 and 1,200,000

16 February 2001 15 February 2001 20 August 2009


Vol 291, Issue 5507, Pages 1145-1434 Vol 409, Issue 6822, Pages 745-964 Vol 460, Pages 1011-1015

Functional classification of human proteins


(many unknown)

Proteomics vs Genomics
 Similarities
- Static picture of dynamic processes
- High throughput analysis
- Technology-driven
- Computation intensive

 Differences
- If we can measure gene expression, why
bother with proteomic?
 protein dynamics and RNA dynamics do
not always correlate
Proteomics Tools Typical Proteomic Studies

 Molecular Biology Tools  Comparing 2 conditions: normal vs. disease


 Identifying every protein in a sample
 Separation & Display Tools
 Protein complexes
 Protein Identification Tools  Identifying unknown protein of interest
 Protein Structure Tools  Post-translational modification
 Quantify proteins or peptides

Typical Proteomic Studies


Typical Proteomic Studies Workflows and Instrumentation
Typical Proteomic Studies Application in Proteomics
Workflows and Instrumentation
Clinically, the decompensated stage of heart
failure is usually preceded by chronic,
compensatory cardiac hypertrophy.

 The molecular mechanisms that precipitate


the transition from stable hypertrophy to failure
remain largely unknown

Application in Proteomics Application in Proteomics


A Proteomic Approach to Determine the Alterations in A Proteomic Approach to Determine the Alterations in
Protein Expression Protein Expression

Separation
Application in Proteomics Application in Proteomics
A Proteomic Approach to Determine the Alterations in A Proteomic Approach to Determine the Alterations in
Protein Expression Protein Expression
Identification Identification

cTnT Degradation in LVH/HF

Application in Proteomics Application in Proteomics


A Proteomic Approach to Determine the Alterations in A Proteomic Approach to Determine the Alterations in
Protein Expression Protein Expression
Data Interpretation Advanced Applications of MS

1. Using a proteomic approach, altered 1. Phosphorylation site identification


expression of cTnT, an important regulatory 2. Protein complex identification
contractile protein, in HF compared to LVH 3. Protein isoform problem
4. Peptide/Protein quantification
2. Data indicate cTnT is degraded near the C-
terminus in HF, but not in LVH
Post-translational Modifications (PTM)
- Phosphorylation 80
- Acetylation 40
- Methylation 14
- Nitrosylation 45
- Oxidation 16
Challenges in Proteomics Proteomics
Take Home Messages

 Complexity – some proteins have >1000


variants  Various protein chemical techniques
 Need for a general technology for targeted  Targeted & Global Studies
manipulation of gene expression  Unique insights into complex systems
 Limited throughput of todays proteomic
 Mass Spectrometry is a key technology
platforms
for proteomics
 Lack of general technique for absolute
quantitation of proteins