lecture 9. functional genomics at the protein level: proteomics
TRANSCRIPT
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Lecture 9. Functional Genomicsat the Protein Level: Proteomics
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Functional Genomics: Development and Application of Genome-Wide Experimental Approaches to Assess Gene Function by making use of the information and reagents provided by Structural Genomics
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Goals of Functional Genomics:1)DNA2)RNA3) Protein4) Whole organism5) Society
Lander, E. 1996. The New Genomics: Global Views of Biology. Science 274: 536-539.
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Goals of Proteomicsa) monitoring the expression and modification state of all proteins in a cell; comparison of proteomesbetween cells
b i) systematic catalogs of all protein:protein interactions (e.g., yeast two hybrid interactions; protein chips; co--IP; affinity chromatography; resolution of complex mixtures purified from cells) ii) systematic catalogs of biochemical interactions, eg., protein kinase/substrate interactions
c) application of structural biochemistry to genomics: classifying proteins by their shapes
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Why proteomics?
1) Expression of many proteins does not correlatewith mRNA levels (in yeast estimated that for50-60% of proteins there is not a linear correlationbetween protein expression and mRNA expression)
2) Many proteins are expressed in an inactive formand only activated post-translationally
3) Many proteins function as part of a complex, andmRNA expression will not reveal these interactions
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Mass Spectrometry is the Key Technology for Proteomics
Separates Ions in the Gas Phase Based on mass/charge (m/z) ratio
See this web site for detailed information about Mass Spec:
http://info.med.yale.edu/wmkeck/
1. Determining the Proteome of Cells
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In gel
Peptide “fingerprint”
OR
2D Gel Electrophoresis is Coupled with One of Two Types of MS
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Peptide Mixture
MALDI (Matrix Assisted Laser Desorption Ionization) Mass Spec
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The Actual Mass Spectrum is Compared to Theoretical Mass Spectrum Predicted for All Proteins in The Genome
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Peptide Separation
Alternative: HPLC Separation of Peptides
Compare peptide mass + “sequence tags” to all possible patterns in the database
Nanospray Tandem Mass Spec (MS/MS)
MS/MS Techniques Can also be Used to Detect Protein Modification(e.g., phosphorylation. acetylation, etc.)
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PI
MW
The 2D Gel is limiting: Rare Proteins or Proteins with ExtremePI (or MW) may not be detected
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Comparing the Proteomes of cells Under Different Conditions
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2. Cataloging Protein:Protein Interactions
a. Mass Spec to Determine Protein:Protein Interactions
Wave of the Future:Determining the Identity of all Proteins inComplicated Mixtures
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B. High Throughput Assays to Determine Protein:Protein Interaction I:Yeast Two-Hybrid Assay
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B. High Throughput Assays to Determine Protein:Protein Interaction II:
Protein Chips
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Comparison of Different Media for Protein Chips
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Protein Chips can also be Used to Study Biochemical reactions:e.g., to Idenitfy Protein Kinase Substrates.
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C. Fluorescent Resonance Energy Transfer (FRET) to Study Protein:Protein Interactions Inside Cells
GFPFluorescence
High Throughput Assays can be Developed FRET
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3. Application of Structural Biology to Genomics:Predicting Protein Function Based on Protein Shape
Conserved Primary Sequences in Protein Family=
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Conserved Secondary Structure=
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CONSERVED TERTIARY PROTEIN FOLDING