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Introduction to Proteomics

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Proteomics Session 4 From protein to proteomics Proteins are the molecule tools for most cellular functions Current -omics Many facts of proteomics Proteomic analysis ... – PowerPoint PPT presentation

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Title: Introduction to Proteomics


1
Proteomics
Session 4 From protein to proteomics
2
Proteins are the molecule tools for most
cellular functions
3
Current -omics
4
Many facts of proteomics
  • Proteomic analysis (global profiling proteomics)
  • Large scale identification and characterization
    of proteins and all their properties.
  • Expression proteomics
  • Comparison between normal and disease. Also
    called differential display proteomics.
  • Cell mapping proteomics
  • Study of protein-protein interaction.

5
Expression proteomics
Example 1, Wild type C57/Black/6J murine colonic
crypt.
WT
EXP
Global profiling proteomics
6
Is cell mapping the destination?
Is Protein-protein interaction study enough to
explain the whole proteomics?
NO
  1. Oversimplify the protein-protein interaction.
  2. Non-specific binding in protein complex mining,
    including affinity capturing, Y2H etc.
  3. Biological important protein-protein interactions
    require that the interacting partners be in
    specific protein states.

7
Physical vs. biological interaction
In other words, proteomics is dynamics
8
Face the real world, what effect protein?
  1. Covalent modification
  2. Cellular localization
  3. Presence of ligands
  4. Alternate splicing
  5. Proteolytic cleavage
  6. Oligomer state
  7. Protein conformation
  8. ..

They are all elements constituted systems biology
9
1. Covalent modification
  1. Phosphorylation, often involve in regulation
  2. Glycosylation, common outside cells
  3. Lipidation, attach to membrane
  4. Nitrosylation, also involve in regulation
  5. Acetylation, common on first aa.
  6. Ubiquitination, for degradation

10
Example nitrosylation
  1. cell signaling,
  2. general cellular regulation processes,
  3. involved in certain signal transduction pathways,
    (cancers and other cell abnormalities)

Proc. Natl. Acad. Sci. USA. 2000 December 5 97
(25) 1354313548
11
2. Cellular locolization
  1. Nuclear protein
  2. Cytosolic protein
  3. Plasma membrane protein
  4. Mitochodria protein
  5. Endoplasmic reticulum protein

Sub-proteome
12
3. Presence of ligand
  1. Heme heme oxygenase 1. hemogolbin,myoglobin
  2. Metal ion Zinc ion in Carbonic anhydrase or
    carboxypeptidase Y
  3. ATP/ADP actin polymerization
  4. GTP/GDP translation factors and signal
    transduction related protein.

13
Example actin polymerization
G-actin (globular actin), with bound ATP, can
polymerize to form F-actin (filamentous).
F-actin may hydrolyze bound ATP ? ADP Pi
release Pi. ADP release from the filament does
not occur because the cleft opening is
blocked. ADP/ATP exchange G-actin can release
ADP bind ATP, which is usually present at
higher concentration than ADP in the cytosol.
14
Example On-Off Switches GTP-Binding Proteins
Ras (p21ras) is a good example of this type of
switch. Ras is a small (21 kDa) monomeric protein
that binds GTP or GDP and has intrinsic GTPase
activity
This causes exchange of bound GDP for GTP
Guanine nucleotide exchange factor interacts with
ras
p21ras
Activated ras interacts with and activates the
next component in the signalling pathway
On
GTP
GDP
ACTIVE
INACTIVE
Pi
Ras GTPase stimulated by association with
GTPase-activating protein (GAP)
Off
Intrinsic GTPase activity hydrolyses GTP to GDP
and Pi
15
4. Alternate splicing
  • Different forms of a protein molecule might
    result from alternate splicing of the gene
    product.

addition of cap, polyA tail
- Poly A Tail
5 Cap-
Splicing
- Poly A Tail
5 Cap-
Mature mRNA
16
Types of mRNA splicing
  • Types I II self-splicing of catalytic RNA
    sequences (ribozymes)
  • In prokaryotes and eukaryotes
  • Type III occurs in a protein - RNA complex -
    Responsible for nearly all splicing (spliceosome)
  • Only in eukaryotes

17
Diagram of alternate splicing
  • Alternate splicing can be regulated differential
    splicing can occur at different times during
    development and in different cell types

GENE (hnRNA)
2
3
4
1
1
2
3
4
2 Different mRNA products
1
3
4
18
Example rat ?-tropomyosin
19
5. Proteolytic cleavage
  • Blood clotting or catabolic digestion pepsin,
    rennin, trypsin, chymotrypsin and
    carboxypeptidase secreted as zymogen.
  • Apoptosis eg. Caspase.
  • Viral protein cleavage of precursor viral
    proteins to functional units

20
Example Apoptosis
signal tranduction pathway regulated by caspase
21
Whats caspase?
  • Cysteine proteases that cleave at aspartic acid
    residues (-Asp-Glu-Xxx-Asp-Xxx-)
  • Caspases are constitutively expressed in inactive
    zymogen form as procaspases
  • Activated by specific proteolysis - often by
    another caspase

22
Caspase 3 dependent gene inactivation
23
6. Oligomeric state
  • A protein molecule may exist in a multiprotein
    complex or as a homodimer of homo-oligomer.
  • 78 of yeast protein occurs in complex. (Gavin A,
    et al., Nature (2002) 415141-147)

24
Gavin A, et al., Nature (2002) 415141-147
25
7. Protein conformation
  • protein function is often regulated by allosteric
    regulation. Eg. Feedback control.
  • Allostetic effector usual bears no structure
    resemblance to substrate of target protein.
  • Examples hexokinase, phosphofructokinase-1,
    pyruvate kinase in glycolysis

26
The other type regulation intrasteric regulation
IARS intrasteric autoregulatory sequence IRS
intrasteric regulatory sequence
Pseudosubstrate
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