Chapter 8 Proteomics

暨南大學資訊工程學系 黃光璿 2004/06/07 1

  proteome  the sum total of an organism’s proteins genome  the sum total of an organism’s genetic material 2

8.1 From Genomes to Proteomes

We want to know   what proteins are present in cells; what those proteins do and how they function.

However, it’s not easy.

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Why?

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The longevity ( different.

壽命 ) of an mRNA and the protein it codes for are very Many proteins are extensively modified after translation.

Many proteins are not functionally relevant until they are assembled into larger complexes or delivered to an appropriate location.

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   Proteins require more careful handling than DNA.

Function may change.

  Protein identification requires mass spectrometric analysis specific antibodies.

Obtaining large numbers of protein molecules requires chemical isolation for living cells.

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8.2 Protein Classification

Based on  protein function  six categories  evolutionary history & structural similarity  1000 homologous families 6

8.2.1 Enzyme Nomenclature

 Started at 1950s

International Union of Biochemistry and Molecular Biology

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8.2.2 Family and Superfamily

   Modern-day proteins may be derived from ~ 1000 original proteins.

folds  superfamilies  families databases  SCOP , CATH , DALI 8

   fold  the same major secondary structure & topological connections superfamily  probable evolutionary relationships family  clear evolutionary relationships 9

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8.3 Experimental Techniques

  2D Electrophoresis Mass Spectrometry 12

2D Electrophoresis

liver http://tw.expasy.org/cgi-bin/map1 kidney

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Problems    tens of thousand v.s. thousands under presentation of membrane-bound proteins difficult to determine exactly which protein is represented 16

8.3.2 Mass Spectrometry

2D  mass spectrometry, for identification 17

8.3.3 Protein Microarrays

 Use antibodies as probes.

Problems  Single proteins will interact with multiple probes.

  The binding kinetics of each probe are different.

Proteins are sensitive to their environment.

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8.4 Inhibitors and Drug Design

   development & testing of a new drug  ~ 15 years, US$ 700 million discovery  target identification  lead discovery & optimization   toxicology ( 毒理學 ) pharmacokinetics testing 19

 HIV protease  has an active site;  cuts a single, large polypeptide chain into many proteins.

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8.5 Ligand Screening

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8.5.1 Ligand Docking

Determine how two molecules of known structure will interact.

Three issues:   Identify the energy of a particular molecular conformations.

Search for the conformation that minimizes the free energy.

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 How to deal with flexibility in both the protein and the putative ligand.

 Lock and key approaches  rigid protein structure, flexible ligand structure  induced fit docking  flexible in both protein and ligand 23

 Softwares  AutoDock  FTDock     DOCK Hammerhead Gold FlexX 24

8.5.2 Database Screening

 Primary consideration  complete and accurate search  with a reasonable computational complexity   SLIDE Fig. 8.4

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8.6 X-Ray Crystal Structures

   W. C. Roentgen (1895) discovered X rays.

M. von Laue (1912) discovered crystals diffract X-rays.

D. Hodgkin, etc. (1950s), crystallized complex organic molecules and determined their structures.

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 grow a crystal of the protein 28

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 File formats  PDB formatted text  mmCIF (MacroMolecular Crystallographic Information File) 31

 databases & resources  PDB  PIR  ExPASy 32

        Visualizing Tools Fig. 8.8

RasMol Swiss PDB viewer VMD (Visual Molecular Dynamics) Spock Protein explorer DINO 33

8.7 NMR Structures

  ~ 200 amino acids the structures determined are not unique 34

8.8 Empirical Methods and Prediction Techniques      Example: Fig. 8.9

extracting features learning, training testing 35

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8.9 Post-Translational Modification Prediction    Remove segments of a protein.

Covalently attach sugars, phosphates, or sulfate groups into surface residues.

Cross-link residues within a protein (disulfide bond).

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8.9.1 Protein Sorting

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 associated with membranes  not associated with membranes Table 8.3 (Case 2) 39

  PSORT : nearest neighbor classifier  Prediction of protein subcellular localization SignalP : artificial neural networks  Prediction of signal peptide cleavage sites 40

8.9.2 Proteolytic Cleavage

   chymotrypsin  cleaves polypeptides on the C-terminal side of bulky and aromatic residues trypsin  cleaves on the carboxyl side elastase  cleaves on the C-terminal side of small residues 41

 Prediction  proteasomes, > 98%, by neural network 42

8.9.3 Glycosylation

 The process of covalently linking an oligosaccharide to the side chain of a protein surface residue ( 科學人 )   N-linked, 75% O-linked, 85% by neural network 43

8.9.4 Phosphorylation

  kinases : add phosphatases : remove  signal  NetPhos , > 70%, neural network 44

參考資料及圖片出處

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Fundamental Concepts of Bioinformatics Dan E. Krane and Michael L. Raymer, Benjamin/Cummings, 2003. Merrian-Webster Dictionary 45