1. Novel Proteomics Techniques
2017/4/28
蛋白質體學
Proteomics 2010
Novel Proteomics Techniques
陳威戎
純化酵素是一件非常基本的工作，很多重要的研究，都脫不開酵素的純化工作。而大多數酵素的純化，基本上也脫不開一些最基本的原則。
首先，建立一個完善的酵素實驗室是很必要的；我們把許多實驗室內的運作細節一一交代，期望同學能認知這些經驗，確實接收並養成習慣，且期望應用到將來每個人的研究工作上。
最先遇到，但是最容易被忽視的步驟，就是材料處理及總蛋白質的抽取。將提醒你小心選擇採料的種類、時期、部位等，並選擇一個良好的粗抽取方式，以便有一個良好的開始。

2. Mass spectrometer schematic

3. Matrix Assisted Laser Desorption Ionization (MALDI)
Animation

4. Electrospray ionization (ESI)
Generates ions directly from acidic solution
The production of ions by evaporation of charged droplets obtained through spraying or bubbling, has been known about for centuries, but it was only fairly recently discovered that these ions may hold more than one charge4. A model for ion formation in ESI, containing the commonly accepted themes, is described below5:
Large charged droplets are produced by 'pneumatic nebulization'; i.e. the forcing of the analyte solution through a needle (see figure), at the end of which is applied a potential - the potential used is sufficiently high to disperse the emerging solution into a very fine spray of charged droplets all at the same polarity. The solvent evaporates away, shrinking the droplet size and increasing the charge concentration at the droplet's surface. Eventually, at the Rayleigh limit, Coulombic repulsion overcomes the droplet's surface tension and the droplet explodes. This 'Coulombic explosion' forms a series of smaller, lower charged droplets. The process of shrinking followed by explosion is repeated until individually charged 'naked' analyte ions are formed. The charges are statistically distributed amongst the analyte's available charge sites, leading to the possible formation of multiply charged ions under the correct conditions. Increasing the rate of solvent evaporation, by introducing a drying gas flow counter current to the sprayed ions (see figure), increases the extent of multiple-charging. Decreasing the capillary diameter and lowering the analyte solution flow rate i.e. in nanospray ionization, will create ions with higher m/z ratios (i.e. it is a softer ionization technique) than those produced by 'conventional' ESI and are of much more use in the field of bioanalysis.

5. Novel Proteomics Techniques
2017/4/28
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

6. Novel Proteomics Techniques
2017/4/28
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

7. SELDI protein chip Surface Enchanced Laser Desorption / Ionization
2017/4/28
Surface Enchanced Laser Desorption / Ionization
Protein chips MALDI-TOF based instrument
表面 修飾 雷射 脫附 離子化
Different chromatographic surfaces
(e. g. anion exchanger, cation exchanger, reverse phase)

8. SELDI ProteinChip Chemical Surfaces Biochemical Surfaces
Hydrophobic Ionic IMAC Mixed
Biochemical Surfaces
    
   
Antibody DNA Enzyme Receptor Drug

10. SELDI ProteinChip (Ciphergen)
2017/4/28
SELDI ProteinChip (Ciphergen)
SELDI – surface enhanced laser desorption/ ionization
Five surface types of 8 array ProteinChips®
The Protein Chip Arrays distinguish this technology from other mass spectrometry-based systems. The Arrays provide a variety of surface chemistries for researchers to optimize protein capture and analysis. The chemistries include classical chromatographic surfaces such as hydrophobic for reversed-phase capture, cation-and anion exchange surfaced, immobilized metal affinity capture (IMAC) for capturing metal-binding proteins, and pre-activated surfaces to investigate antibody-antigen, DNA-protein, receptor-ligand, etc.
Protein chips

11. SELDI
2017/4/28

12. SELDI ProteinChip- suitable for biomarker discovery

13. Novel Proteomics Techniques
2017/4/28
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

14. 2. Multiple Dimensional Liquid Chromatography, MDLC

15. 2. Multiple Dimensional Liquid Chromatography, MDLC

16. Novel Proteomics Techniques
2017/4/28
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

17. 3. Detection of dynamic changes in tissue/cells
Stable-isotope labeling with amino acids in cell culture (SILAC)

18. 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 stable-isotope incorporation is not known until the sequence is determined.

19. Isotope Coded Affinity Tags Technology, ICAT

20. Isotope Coded Affinity Tags Technology, ICAT

21. Isotope Coded Affinity Tags Technology, ICAT

22. Advantages: Disadvantages:
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.

23. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

24. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

25. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

26. isobaric Tag for Relative and Absolute Quantitation , iTRAQ

27. isobaric Tag for Relative and Absolute Quantitation , iTRAQ

28. 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.

29. 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.

30. Novel Proteomics Techniques
2017/4/28
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

31. 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.

32. 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

33. Sample A pre-labelled with dye A Sample B pre-labelled with dye B
4. Difference Gel Electrophoresis (DIGE)
Sample A pre-labelled with dye A
Sample B pre-labelled with dye B
Differential View
Cy5
Cy3
Cy3/cy5 staining
Cy5
Cy3
Cy5/Cy3 staining
... 1 Gel, 2 Samples, 2 Dyes: More Data Reliability with Higher Speed!!

34. 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 differential image analysis software.

35. 4. Difference Gel Electrophoresis (DIGE)
Cyanine Dyes (Cy2, Cy3, and Cy5)
Fluorophore
Absorption Peak (nm)
Emission Peak (nm)
Cyanine, Cy2
492
510
Fluorescein, FITC
520
Indocarbocyanine, Cy3
550
570
Tetramethyl Rhodamine, TRITC
Indodicarbocyanine, Cy5
650
670

36. 4. Difference Gel Electrophoresis (DIGE)
Cyanine Dyes (Cy2, Cy3, and Cy5)
Excitation
Emission

37. 4. Difference Gel Electrophoresis (DIGE)
IEF+SDS PAGE
Control
（untreated）
Cy3
（Cy5）
Mix
DeCyder DIA
Treated
Sample
Cy5
（Cy3）
Internal
standard
Cy2

38. 4. Difference Gel Electrophoresis (DIGE)

39. 4. Difference Gel Electrophoresis (DIGE)

40. 4. Difference Gel Electrophoresis (DIGE)