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蛋白质组学 PowerPoint PPT Presentation


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蛋白质组学. 背 景. 基因数量有限性和基因结构的相对稳定性 VS 生命现象的复杂性和多变性. 从 genomic 到 proteome. 对蛋白质的数量、结构、性质、相互关系和生物学功能进行全面深入的研究已成为生命科学研究的迫切需要和重要任务。. The era of ‘ omics ’ -based science Genomics (基因组学) Post-genomic science (后基因组时代) Functional genomics (功能基因组学) Transcriptomics ( 转录组学) Proteomics (蛋白质组学)

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蛋白质组学

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VS

genomicproteome


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  • The era of omics-based science

    • Genomics

    • Post-genomic science

      • Functional genomics

        • Transcriptomics

        • Proteomics

        • Metabolomics

      • Structural genomics

        • Aim - 3D structures of all protein folds !


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proteomePROTEins + genOMEProteins expressed by a genome

1994WilliamsWilkins


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Proteomics

(proteomics)


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Proteomics

The study of global changes in protein expression

The systematic study of protein-protein interactions through the isolation of protein complexes


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  • The study of proteins expressed by genomes

  • Completion of the sequencing of the 1st draft of human genome indicates there are approximately 250,000 proteins in the human genome

  • Only 2-5% of proteins in human genome have been identified


Functional proteomics

(functional proteomics)


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1996Australia Proteome Analysis FacilityAPAF


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NCI1 000

NCIFDA


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Celera


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1997

1998

19991


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1998


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2003CHHUPO200392004820058200410


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  • 973863


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Laser capture microdissection lcm

(laser capture microdissectionLCM)


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  • two-dimensional electrophoresis2-DE)


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  • 10100 kD


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2-DE


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liquid chromatographyLC

capillary electrophoresisCE


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  • 30~100ng2040100

  • PAGE8~10ng

  • PVDF/


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  • PAGE

  • 5~15minEDTATris/


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  • PVDF

  • PAGE


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  • SYPRO Red Orange Ruby

  • SDS-PAGE30~60min2~10ng300nm3

  • SAGE

  • Tris/Edman


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  • Tween-20

  • SYPRO Ruby


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  • 2-DE


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  • :

    MelanieII (BioRad),

    PD Quest (BioRad),

    Phoretix 2D Full, (Amersham Pharmacia Biotech)

    ImageMaster 2d Platinum


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m/z

m/z


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20

2040

2060

2080

J.J. Thomson

-

-


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Sir Joseph John Thomson - 1906Frederick Soddy - 1921Francis William Aston - 1922Hans G. Dehmelt and Wolfgang Paul - 1989Robert F. Curl Jr. & Sir Harold W. Kroto & Richard E. Smalley - 1996C60-C80John Fenn and Koichi Tanaka with Kurt Wuthrich - 2002


Ion source

Ion source

  • (Electron Ionization EI)

  • (Chemical Ionization , CI )

  • Fast Atomic bombardment, FAB

  • (Electron spray IonizationESI)

  • (Atmospheric pressure chemical Ionization, APCI)

  • Atmospheric pressure photoionization, APPI

  • MALDI-TOF


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100,000

10,000

ESI/FAB

MW

APPI

1000

APCI

100

EI/CI

10

Polarity

Very Polar

Nonpolar


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  • 80pmol

  • 10,000


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(Sequence database search):

(De novo interpretation)


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  • , ,

  • , , .

  • , , ,


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SWISS-PROT


Mascot search engine

Mascot Search Engine


Mascot ms ms ions search

Mascot MS/MS Ions Search


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1yeast two-hybrid system

  • 1989Field Song


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GAL4

  • NNLSGAL1(UASG)

  • CGAL1


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  • GAL4 BD AD


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  • cDNA


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1.

2.

3.


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2


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1

  • SDSMDLC-ESI-MS/MS


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  • bait


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2

  • immuno-precipitationIP


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p53

  • p53HNE1HNE2

  • SDS-PAGE

  • p53LC-ESI-MS/MS


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A.

B.

C.


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3

  • TAP Tag


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IgG

TEV


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EGTA


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3. protein chipsprotein array


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