Download Novel Proteomics Techniques

蛋白質體學
Proteomics 2010
Novel Proteomics Techniques
陳威戎
2010. 11. 23
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Mass spectrometer schematic
Matrix Assisted Laser Desorption
Ionization (MALDI)
Animation
Electrospray ionization (ESI)
•
Generates ions directly from acidic solution
Novel Proteomics Techniques
1. SELDI protein chips (Ciphergen)
2. Multiple Dimensional Liquid Chromatography, MDLC
3. Detect Dynamic Changes in Tissue/Cells
(1) Stable-Isotope Labeling
(2) Isotope Coded Affinity Tags Technology, ICAT
(3) Isobaric Tag for Relative and Absolute
Quantitation, iTRAQ
4. Difference Gel Electrophoresis, DIGE
Novel Proteomics Techniques
1. SELDI protein chips (Ciphergen)
2. Multiple Dimensional Liquid Chromatography, MDLC
3. Detect Dynamic Changes in Tissue/Cells
(1) Stable-Isotope Labeling
(2) Isotope Coded Affinity Tags Technology, ICAT
(3) Isobaric Tag for Relative and Absolute Quantitation,
iTRAQ
4. Difference Gel Electrophoresis, DIGE
SELDI protein chip
Surface Enchanced Laser Desorption / Ionization
Protein chips
+
MALDI-TOF based instrument
Different chromatographic surfaces
(e. g. anion exchanger, cation exchanger, reverse phase)
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SELDI ProteinChip
• Chemical Surfaces
Hydrophobic
Ionic
IMAC-3
Mixed
• Biochemical Surfaces
Antibody
DNA
Enzyme
Receptor
Drug8
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SELDI ProteinChip (Ciphergen)
SELDI – surface enhanced laser desorption/ ionization
Protein chips
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SELDI
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SELDI ProteinChip- suitable for biomarker
discovery
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Novel Proteomics Techniques
1. Protein Arrays ; SELDI protein chips (Ciphergen)
2. Multiple Dimensional Liquid Chromatography, MDLC
3. Detect Dynamic Changes in Tissue/Cells
(1) Stable-Isotope Labeling
(2) Isotope Coded Affinity Tags Technology, ICAT
(3) Isobaric Tag for Relative and Absolute Quantitation,
iTRAQ
4. Difference Gel Electrophoresis, DIGE
2. Multiple Dimensional Liquid Chromatography, MDLC
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2. Multiple Dimensional Liquid Chromatography, MDLC
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Novel Proteomics Techniques
1. Protein Arrays ; SELDI protein chips (Ciphergen)
2. Multiple Dimensional Liquid Chromatography, MDLC
3. Detect Dynamic Changes in Tissue/Cells
(1) Stable-Isotope Labeling
(2) Isotope Coded Affinity Tags Technology, ICAT
(3) Isobaric Tag for Relative and Absolute Quantitation,
iTRAQ
4. Difference Gel Electrophoresis, DIGE
5. Analysis of protein-protein interactions
(1) Complex isolation
- Coimmunoprecipitation ; Affinity purification
(2) Finding partners
- Yeast two-hybrid ; Phage display system
6. Post-translational modifications, PTM
3. Detection of dynamic
changes in tissue/cells
 Stable-isotope labeling
with amino acids in cell
culture (SILAC)
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Drawbacks:
 The method does not allow for the analysis of
proteins directly from tissue.
 The stable-isotope enriched media are costly and
may themselves affect cellular growth and protein
production.
 The increase in nominal mass because of stableisotope incorporation is not known until the sequence
is determined.
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 Isotope Coded Affinity Tags Technology, ICAT
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 Isotope Coded Affinity Tags Technology, ICAT
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 Isotope Coded Affinity Tags Technology, ICAT
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Advantages:
 The method is compatible with any amount of protein harvested
from bodily fluids, cells or tissues under any growth conditions.
 The alkylation reaction is highly specific and occurs in the
presence of salts, detergents, and stabilizers (e.g. SDS, urea,
guanidine-HCl).
 The complexity of the peptide mixture is reduced by isolating
only cysteine-containing peptides.
 The ICAT strategy permits almost any type of biochemical,
immunological, or physical fractionation, which makes it
compatible with the analysis of low-abundance proteins.
Disadvantages:
 The size of the ICAT label (~500 Da) is a relatively large
modification.
 The method fails for proteins that contain no cysteines.
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 isobaric Tag for Relative and Absolute
Quantitation , iTRAQ
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 isobaric Tag for Relative and Absolute
Quantitation , iTRAQ
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 isobaric Tag for Relative and Absolute
Quantitation , iTRAQ
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isobaric Tag for Relative and Absolute
Quantitation , iTRAQ
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 isobaric Tag for Relative and Absolute
Quantitation , iTRAQ
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 isobaric Tag for Relative and Absolute
Quantitation , iTRAQ
 Non-gel based technique
 Uses isotope coded covalent tags.
 Covalent labeling of the N-terminus and sidechain amines
of pepitdes from protein digestions with tags of varying mass.
 Simultaneously identify and quantify proteins from different
sources (multiple sample) in one single experiment.
 Increases confidence in identification and quantitation from
MS/MS spectra by tagging multiple peptides per protein.
 Increases throughput and confidence in results for protein
biomarker discovery studies.
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 isobaric Tag for Relative and Absolute
Quantitation , iTRAQ
 Two mainly used reagents:
4-plex and 8-plex.
 Pooled and fractionated by
nano liquid chromatography and
analyzed by tandem mass
spectrometry (MS/MS)
 Expands proteome coverage by labeling all peptides,
including those with post-translational modifications (PTMs).
 Offers a simple workflow without sample fractionation for
reduced-complexity samples, such as affinity pull-downs.
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Novel Proteomics Techniques
1. Protein Arrays ; SELDI protein chips (Ciphergen)
2. Multiple Dimensional Liquid Chromatography, MDLC
3. Detect Dynamic Changes in Tissue/Cells
(1) Stable-Isotope Labeling
(2) Isotope Coded Affinity Tags Technology, ICAT
(3) Isobaric Tag for Relative and Absolute Quantitation,
iTRAQ
4. Difference Gel Electrophoresis, DIGE
4. Difference Gel Electrophoresis (DIGE)
 Allows the separation of treated (or diseased) and
untreated (or control) samples in a single physical gel.
 Quick comparison in the differences of the protein profiles
of each sample by overlaying the unwrapped maps of treated
and untreated samples. It is possible to see which proteins are
shared by both, which are present in one sample but not in the
other.
 In a DIGE system, proteins are pre-labelled with
fluorescent CyDyes™ such as Cy3, and Cy5 prior to
electrophoretic separations. Labelled samples are then mixed
before isoelectric focusing, and resolved on the same 2D gel.
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4. Difference Gel Electrophoresis (DIGE)
Key benefits:
 More confidence- reflects true biological outcomes and is
not due to the technical variation
 Less gels- saves time by reducing the large number of
replicates that are used in the conventional, single stain 2D
gel method
 High accuracy- no false negative and no false positive
 Quantitative data
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4. Difference Gel Electrophoresis (DIGE)
Sample A pre-labelled with dye A
Cy3
Cy5
Sample B pre-labelled with dye B
Cy5
Cy3
Differential View
Cy3/cy5
staining
Cy5/Cy3
staining
... 1 Gel, 2 Samples, 2 Dyes: More Data Reliability with Higher Speed!!
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4. Difference Gel Electrophoresis (DIGE)
 In a new DIGE system, proteins are pre-labelled with
fluorescent CyDyes™ such as Cy2, Cy3, and Cy5 prior to
electrophoretic separations. Labelled samples are then mixed
before isoelectric focusing, and resolved on the same 2D gel.
 Cy2 dye is used to label an internal standard, which
consists of a pooled sample comprising of equal amounts of
each of the samples to be compared. This allows both inter
and intra gel matching, and is used in the standardization of
spot volumes in different gels. Spot volumes are expressed as
a ratio to the internal standard.
 Images of each dye are acquired with various lasers using
a variable mode imager and images are analyzed with
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differential image analysis software.
4. Difference Gel Electrophoresis (DIGE)
 Cyanine Dyes (Cy2, Cy3, and Cy5)
Fluorophore
Absorption Peak
(nm)
Emission Peak
(nm)
Cyanine, Cy2
492
510
Fluorescein, FITC
492
520
Indocarbocyanine, Cy3
550
570
Tetramethyl Rhodamine,
TRITC
550
570
Indodicarbocyanine, Cy5
650
670
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4. Difference Gel Electrophoresis (DIGE)
 Cyanine Dyes (Cy2, Cy3, and Cy5)
Excitation
Emission
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4. Difference Gel Electrophoresis (DIGE)
IEF+SDS PAGE
Control
Cy3
（untreated） （Cy5）
Mix
Treated
Sample
Internal
standard
DeCyder DIA
Cy5
（Cy3）
Cy2
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4. Difference Gel Electrophoresis (DIGE)
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4. Difference Gel Electrophoresis (DIGE)
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4. Difference Gel Electrophoresis (DIGE)
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