1. Novel Proteomics Techniques and Bioinformatics
2018/5/2
蛋白質體學
Proteomics 2016
Novel Proteomics Techniques and Bioinformatics
陳威戎

2. Novel Proteomics Techniques
2018/5/2
1. SELDI protein chips
2. Multiple Dimensional Liquid Chromatography, MDLC
3. Quantitative Proteomics
(1) Stable-Isotope Labeling, SILAC
(2) Isotope Coded Affinity Tags Technology, ICAT
(3) Isobaric Tag for Relative and Absolute Quantitation, iTRAQ
4. Bioinformatics for proteomics

3. 1. SELDI-TOF MS (Ciphergen)
2018/5/2
Surface Enchanced Laser Desorption / Ionization
表面 修飾 雷射 脫附 離子化

4. SELDI protein chips Surface Enchanced Laser Desorption / Ionization
2018/5/2
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)

5. SELDI protein chips Chemical Surfaces Biochemical Surfaces
Hydrophobic Ionic IMAC Mixed
Biochemical Surfaces
    
   
Antibody DNA Enzyme Receptor Drug

7. SELDI protein chips Protein chips
2018/5/2
SELDI protein chips
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

8. SELDI
2018/5/2

9. SELDI Protein Chip- suitable for biomarker discovery

10. SELDI-TOF, Biomarker discovery
Animation

11. Comparative MS profiles among patients and normal control

12. Comparative MS profiles among patients and normal control

13. 2. Multiple Dimensional Liquid Chromatography, MDLC

14. 2. Multiple Dimensional Liquid Chromatography, MDLC

15. 2. Multiple Dimensional Liquid Chromatography, MDLC

16. 2’. Multi-Dimensional LC-MS/MS

17. 2’. Multi-Dimensional LC-MS/MS

18. 3. Quantitative Proteomics: detection of dynamic changes in tissue/cells
Stable-isotope labeling with amino acids in cell culture (SILAC)

19. Stable-isotope labeling with amino acids in cell culture (SILAC)

20. Stable-isotope labeling with amino acids in cell culture (SILAC)

21. Stable-isotope labeling with amino acids in cell culture (SILAC)

22. Stable-isotope labeling with amino acids in cell culture (SILAC)

23. 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.

24. Isotope Coded Affinity Tags Technology, ICAT

25. Isotope Coded Affinity Tags Technology, ICAT

26. Isotope Coded Affinity Tags Technology, ICAT

27. 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.

28. SILAC vs. ICAT

29. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

30. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

31. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

32. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

33. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

34. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

35. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

36. isobaric Tag for Relative and Absolute
Quantitation , iTRAQ

37. 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.

38. 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.

39. MS vs. MS/MS vs. Reporter ions

40. 4. Bioinformatics for proteomics

41. 4. Bioinformatics for proteomics

42. Mascot Search Results Probability Based Mowse Score Score is -
Search title : SampleSetID: 362, AnalysisID: 567, MaldiWellID:
15790, SpectrumID: 17225, Path= \
Man i
102004
New Analysis 1
Database : NCBInr ( sequences;
residues)
Timestamp : 20 Oct 2004 at 14:52:50 GMT
Top Score : 681 for
gi|180570
, creatine kinase [Homo sapiens]
Probability Based Mowse Score
Score is -
10*Log(P), where P is the probability that the observed match is a random
event.
Protein scores greater than 75 are significant (p<0.05). 42

43. Top hits from Mascot Search – there are multiple accession numbers for the same protein43

44. Creatine kinase B is the highest scoring protein
Match to: gi| ; Score: 681
Creatine kinase - B [Homo sapiens]
Nominal mass (Mr): 42591; Calculated pI value: 5.34
Observed Mass & pI: 43kd,
Sequence Coverage: 46%
1 MPFSNSHNAL KLRFPAEDEF PDLSAHNNHM AKVLTPELYA ELRAKSTPSG
51 FTLDDVIQTG VDNPGHPYIM TVGCVAGDEE SYEVFKDLFD PIIEDRHGGY
101 KPSDEHKTDL NPDNLQGGDD LDPNYVLSSR VRTGRSIRGF CLPPHCSRGE
151 RRAIEKLAVE ALSSLDGDLA GRYYALKSMT EAEQQQLIDD HFLFDKPVSP
201 LLSASGMARD WPDARGIWHN DNKTFLVWVN EEDHLRVISM QKGGNMKEVF
251 TRFCTGLTQI ETLFKSKDYE FMWNPHLGYI LTCPSNLGTG LRAGVHIKLP
301 NLGKHEKFSE VLKRLRLQKR GTGGVDTAAV GGVFDVSNAD RLGFSEVELV
351 QMVVDGVKLL IEMEQRLEQG QAIDDLMPAQ K 44

45. List of useful proteomics websites
Click on each link to get familiar