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阮雪芬 Department of Life Aug 6, 2004

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Presentation on theme: "阮雪芬 Department of Life Aug 6, 2004"— Presentation transcript:

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

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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

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59 Step 1

60 Step 2

61 Step 3

62 Step 3

63 Step 4

64 Step 5

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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

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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

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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)

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92 Fasta Sequence

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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

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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

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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

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105 YinOyang

106 Result


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