Chapter 8Proteomics

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Chapter 8 Proteomics

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• 2004/06/07

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• proteome
• the sum total of an organisms proteins
• genome
• the sum total of an organisms genetic material

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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, its not easy.

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Why?

1. The longevity (??) of an mRNA and the protein it
   codes for are very different.
2. Many proteins are extensively modified after
   translation.
3. 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

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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

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• fold
• the same major secondary structure topological
  connections
• superfamily
• probable evolutionary relationships
• family
• clear evolutionary relationships

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8.3 Experimental Techniques

• 2D Electrophoresis
• Mass Spectrometry

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2D Electrophoresis
liver
kidney
http//tw.expasy.org/cgi-bin/map1
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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

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8.3.2 Mass Spectrometry

• 2D ? mass spectrometry, for identification

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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 (???)
• pharmacokinetics
• testing

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• 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

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• Softwares
• AutoDock
• FTDock
• DOCK
• Hammerhead
• Gold
• FlexX

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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

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• File formats
• PDB formatted text
• mmCIF (MacroMolecular Crystallographic
  Information File)

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• databases resources
• PDB
• PIR
• ExPASy

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• Visualizing Tools
• Fig. 8.8
• RasMol
• Swiss PDB viewer
• VMD (Visual Molecular Dynamics)
• Spock
• Protein explorer
• DINO

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8.7 NMR Structures

• 200 amino acids
• the structures determined are not unique

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8.8 Empirical Methods and Prediction Techniques

• Example
• Fig. 8.9
• extracting features
• learning, training
• testing

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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)

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• PSORT nearest neighbor classifier
• Prediction of protein subcellular localization
• SignalP artificial neural networks
• Prediction of signal peptide cleavage sites

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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

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• Prediction
• proteasomes, gt 98, by neural network

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8.9.3 Glycosylation

• The process of covalently linking an
  oligosaccharide to the side chain of a protein
  surface residue (???)
• N-linked, 75
• O-linked, 85

by neural network
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8.9.4 Phosphorylation

• kinases add
• phosphatases remove
• signal
• NetPhos, gt 70, neural network

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?????????

• Fundamental Concepts of BioinformaticsDan E.
  Krane and Michael L. Raymer, Benjamin/Cummings,
  2003.
• Merrian-Webster Dictionary