1. 阮雪芬 Department of Life Science @NTU Aug 6, 2004
2D Gel Analysis
阮雪芬
Department of Life
Aug 6, 2004

2. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

3. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

4. What Is Proteomes ?

5. Proteomes Gene + Chromosome Genome Protein +Genome Proteome
Proteomes are dynamics
Proteome changes as a function of:
time
development
extracellular condition
intracellular condition

6. Definitions of Proteomics
First coined in 1995 by Wilkins
Be defined as the large-scale characterization of the entire protein complement of a cell line, tissue, or organism.
The study of proteomes
Goal:
-To obtain a more global and integrated view of biology by studying all the proteins of a cell rather than each one individually.

7. Proteomics Origins
In 1975, the introduction of the 2D gel by O’Farrell who began mapping proteins from E. coli.
The first major technology to emerge for the identification of proteins was the sequencing of proteins by Edman degradationpicomole
MS technology has replaced Edman degradation to identify proteinsfemtomole

8. How Proteomics Can Help Drug Development

9. Types of Proteomics and Their Applications to Biology

10. Mechanisms by Which a Single Gene Can Give Rise to Multiple Gene Products
In bacteria, 1 or 2 proteins/gene
In yeast, 3 proteins/gene
In human, 3 or more proteins/gene

11. Two-dimensional Gel Approach
Nature 2000, 405,

12. Image Matching kDa 150 Increase of 50% 70 Decrease of 50% 60 42
Unmatched spots
Matched spots 10 pH
3.5 10

13. Standard Proteome Analysis by 2DE-MS
Mass Fingerprint Searching in
Current Opinion in Chemical Biology 2000, 4:489–494

14. Koichi Tanaka
2002 Nobel Prize:The Origin of Macromolecule Ionization by Laser Irradiation
In 1985 Feb, instead of using Cobalt Ultra Fine Metal Power (UFMP), he mistakenly used a glycerin-UFMP

15. Schematic of Time of Flight Mass Spectrometry
2002 Nobel lecture by Koichi Tanaka

16. Time of Flight Mass Spectrometry

17. How Does a Mass Spectrometer Work?
Sample input
Analyzer
Detector
Ionization

18. Macromolecule Desorption/Ionization using UFMP Glycerin Mixed Matrix
2002 Nobel lecture by Koichi Tanaka
2002 Nobel lecture

19. A Simplified Schematic of the MALDI Ionization Process

20. Mass Spectrometry

21. Peptide Mass Fingerprinting

22. The Information Stored in Genes Is Expressed by a Multistage Process

23. DNA 和蛋白質合成的地方
DNA
Proteins
Sugar Chain

24. Post-translational Modification
DNA and mRNA provide no information concerning the activities and post-translational modifications of proteins.
The number of documented protein co- and post-translational modifications now exceeds 400 (http: / / abrf.org / index-.cfm/dm.home).
The elucidation of protein post-translational modifications is perhaps the most important justification for proteomics as a scientific endeavor.

25. Chemical modification
Covalent linkage of chemical group to amino acid in protein
Several types:
Acetylation (common on first amino acid)
Phosphorylation (often involved in regulation)
Lipidation (attachment to membrane)
Glycosylation (common outside cell)

26. Processing Removal of part of protein by either:
Cleavage (protease cuts protein into smaller pieces)
Splicing (self-removal and rejoining of ends)

27. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

28. Primary Structure Analysis
Object:
To compute the characters of proteins.
-Amino acid composition
-Atomic composition
-pI
-Molecular weight

29. Amino Acid & Atomic Composition
ProtParam

30. Amino Acid & Atomic Composition

31. Amino Acid & Atomic Composition
Amino Acid Composition
Atomic Composition

32. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

33. Computing pI and MW

34. Computing pI and MW

35. Computing pI and MW MW pI

36. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

37. Protein Sequence Searching

38. Protein Sequence Searching

39. Protein Sequence Searching

40. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

41. Sequence Alignment

42. Sequence Alignment Input Query Amino Acid Sequence DNA Sequence blastn
blastx
tblastx
Blastp
tblastn
Compares Against protein Sequence Database
Compares Against Translated Nucleotide Sequence Database
Compares Against protein Sequence Database
Compares Against Translated Nucleotide Sequence Database
Compares Against Nucleotide Sequence Database

43. Sequence Alignment

44. Sequence Alignment

45. Sequence Alignment

46. Sequence Alignment

47. Sequence Alignment

48. Sequence Alignment

49. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

54. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

55. World Wide Web Tools for Searching Databases
Site name
URL
Information available
MOWSE
Peptide mass mapping and sequencing
ProFound
PeptIdent
PepSea
MASCOT
PepFrag
Protein Prospector
FindMod
Posttranslational modification
SEAQUEST
Uninterpreted MS/MS searching
FASTA Search Programs
Protein and nucleotide database searching
Cleaved Radioactivity of
Phosphopeptides
Protein phosphorylation site mapping

56.
Mascot

59. Step 1

60. Step 2

61. Step 3

62. Step 3

63. Step 4

64. Step 5

69. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

71. Step 1

72. Step 2

73. Step 3

74. Step 3

75. Step 4

76. Step 5

77. Step 6

78. Step 7

79. Step 8

80. Step 9

85. For peptide mass fingerprint data

86. pI and MW
Species

87. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

88. Publicly Available PTM Web Resources (I)
Proteomics (2004), 4,

89. Publicly Available PTM Web Resources (II)

92. Fasta Sequence

95. Search Result

96. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

99. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

100. Predotar: A Tool for N-terminal Targeting Sequence
Proteomics (2004) 4,

101. P33316 sequence

103. Outline Introduction to Proteomics Softwares Protein primary structure
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint
Mascot MS
MS/MS
Post-Translational Modification
SignalP
NetPhos
Predotar
YinOYang

105. YinOyang

106. Result